High performance liquid chromatography (hplc) fingerprints of traditional chinese medicine composition for treating nephropathy, construction method and use thereof

Abstract

The application provides a method for constructing a high performance liquid chromatography characteristic map and / or a fingerprint map of a traditional Chinese medicine composition for treating nephropathy, which comprises: (a) preparing a test solution of the traditional Chinese medicine composition for treating nephropathy, and preparing a control solution by taking rosmarinic acid as a control; (b) performing high performance liquid chromatography detection on the test solution and the control solution; wherein the high performance liquid chromatography detection conditions comprise: wavelength of 330 nm, octadecylsilane-bonded silica gel as the filler of a chromatographic column, acetonitrile as a mobile phase A, aqueous phosphoric acid as a mobile phase B, and gradient elution. The application also provides the characteristic map and the fingerprint map constructed according to the above method and uses thereof. The method effectively constructs the characteristic map and the fingerprint map through high performance liquid chromatography, and provides efficient and reliable guarantee for more reliable and more accurate analysis and detection and identification of Xue Nia'an drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical analysis and detection technology. Specifically, the present application relates to a high performance liquid chromatography (HPLC) characteristic map and / or fingerprint map of a traditional Chinese medicine composition for treating kidney disease, in particular, Xue Niaoran tablets or capsules, and a method for constructing the same and uses thereof. BACKGROUND

[0002] Xue Niaoran is a medicine prepared from main raw materials such as Orthosiphon aristatus, Radix Cistanches, Imperata cylindrica, and Phellodendri, etc. It is usually provided in the form of tablets, capsules, etc. and has the effects of clearing heat and removing dampness, and cooling blood and stopping bleeding. It is mainly used for treating acute and chronic glomerulonephritis, pyelonephritis, urinary tract infection, purpura nephritis, and occult nephritis, etc.

[0003] At present, Deng, et al. published a document of "Study on Quality Standard of Xue Niaoran Tablets", which reported the establishment of thin layer identification of Cistanches, Phellodendri, Imperata cylindrica, and Orthosiphon aristatus in Xue Niaoran tablets, and the content determination of berberine hydrochloride and rosmarinic acid, but not the establishment of a fingerprint map or a characteristic map. Zhao, et al. published a technical achievement of "Study on Quality Evaluation Method of Xue Niaoran Capsules and Orthosiphon aristatus", which reported the establishment of thin layer chromatography identification method of Imperata cylindrica, Orthosiphon aristatus, and Radix Cistanches in Xue Niaoran capsules, and the establishment of high performance liquid chromatography method for determining the contents of effective components berberine hydrochloride and rosmarinic acid in Xue Niaoran capsules. Chai, et al. published a document of "Simultaneous Determination of Four Components in Xue Niaoran Capsules by High Performance Liquid Chromatography", which only reported a method of HPLC for multiple evaluation of content determination, but not a method for establishing a fingerprint map. It can be seen that there is still a lack of a characteristic map or a fingerprint map and a method for constructing the same for reliably and accurately identifying, detecting, or reflecting the related substances in a traditional Chinese medicine composition for treating kidney disease, in particular, Xue Niaoran medicine.

[0004] Therefore, based on the deficiencies of the prior art, it is necessary to seek a fingerprint detection method for Xue Niaoran medicine that can better perform quality control, in order to solve the problem that there is no prospective comprehensive detection method for effective components of Xue Niaoran medicine, in particular, Xue Niaoran capsules. SUMMARY

[0005] The first aspect of the present application provides a method for constructing a high performance liquid chromatography (HPLC) characteristic map and / or fingerprint map of a traditional Chinese medicine composition for treating kidney disease. The method effectively constructs the characteristic map and / or fingerprint map by HPLC, which provides a high-efficiency and reliable guarantee for more reliably and accurately analyzing, detecting, and identifying the related substances in a traditional Chinese medicine composition for treating kidney disease, in particular, Xue Niaoran tablets or capsules.

[0006] The second aspect of the present application provides a high performance liquid chromatography (HPLC) characteristic map and / or fingerprint map of a traditional Chinese medicine composition for treating kidney disease, in particular, Xue Niaoran tablets or capsules, obtained according to the above-mentioned method.

[0007] The third aspect of the present application provides a method for detecting a traditional Chinese medicine composition for treating kidney disease, in particular, Xue Nia'an tablets or capsules, namely, the use of the above-mentioned high performance liquid characteristic map and fingerprint map in detecting related substances in the traditional Chinese medicine composition for treating kidney disease, in particular, Xue Nia'an tablets or capsules.

[0008] The above-mentioned aspects of the present application are realized by the following technical solutions:

[0009] In a first aspect, the present application provides a method for constructing a high performance liquid characteristic map and / or a fingerprint map of a traditional Chinese medicine composition for treating kidney disease, comprising:

[0010] (a) preparing a test solution of the traditional Chinese medicine composition for treating kidney disease, and preparing a reference solution with rosmarinic acid as a reference substance; wherein the traditional Chinese medicine composition for treating kidney disease comprises 36-44 parts of Ficus tsiangiana, 54-66 parts of Herba Radix Lycopi, 72-88 parts of Rhizoma Imperatae, and 10-14 parts of Cortex Phellodendri, by weight;

[0011] (b) performing high performance liquid chromatography detection on the test solution and the reference solution; wherein the conditions of the high performance liquid chromatography detection include:

[0012] at a wavelength of 330 nm, with octadecylsilane-bonded silica gel as the filler of the chromatographic column, with acetonitrile as the mobile phase A, with phosphoric acid aqueous solution as the mobile phase B, and with gradient elution, the theoretical plate number calculated based on rosmarinic acid is not less than 400000.

[0013] Preferably, the traditional Chinese medicine composition for treating kidney disease comprises 40 parts of Ficus tsiangiana, 60 parts of Herba Radix Lycopi, 80 parts of Rhizoma Imperatae, and 12 parts of Cortex Phellodendri, by weight.

[0014] In the above-mentioned first aspect, at least the chromatogram with a retention time of 165 min or less is recorded, for example, the chromatogram with a retention time of 165 min or less or 180 min or less can be recorded.

[0015] Preferably, the traditional Chinese medicine composition for treating kidney disease is selected from Xue Nia'an tablets or capsules.

[0016] Preferably, in step (a), the method for preparing the test solution of the traditional Chinese medicine composition for treating kidney disease comprises: dissolving a sample of the traditional Chinese medicine composition for treating kidney disease in a methanol aqueous solution, obtaining after ultrasonic extraction, filtration, and quantification. For example, a sample of the traditional Chinese medicine composition for treating kidney disease is accurately weighed, dissolved in a methanol aqueous solution, ultrasonically treated, taken out and cooled, supplemented with the lost weight with a methanol aqueous solution, shaken, filtered, and the filtrate is taken and quantified to obtain the test solution.

[0017] Preferably, the concentration of the test sample solution is 10 mg / mL to 40 mg / mL, more preferably 30 mg / mL. In the present application, the concentration of the test sample solution can be selected, for example, from 10 mg / mL, 12 mg / mL, 15 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 22 mg / mL, 25 mg / mL, 28 mg / mL, 30 mg / mL, 32 mg / mL, 35 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, and any point value within a range defined by any two of the above point values.

[0018] Preferably, the concentration of the control sample solution is 10 μg / mL to 30 μg / mL, more preferably 20 μg / mL. In the present application, the concentration of the control sample solution can be selected, for example, from 10 μg / mL, 12 μg / mL, 15 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, 22 μg / mL, 25 μg / mL, 28 μg / mL, 30 μg / mL, and any point value within a range defined by any two of the above point values.

[0019] Preferably, the ultrasonic extraction is performed at a power of 400 W to 600 W and a frequency of 30 kHz to 50 kHz for 20 min to 40 min. In the present application, the ultrasonic extraction can be performed, for example, at a power of 400 W, 410 W, 420 W, 430 W, 440 W, 450 W, 470 W, 490 W, 500 W, 510 W, 520 W, 530 W, 540 W, 550 W, 570 W, 590 W, or 600 W and at a frequency of 30 kHz, 35 kHz, 40 kHz, 45 kHz, or 50 kHz for 20 min, 25 min, 30 min, 35 min, or 40 min.

[0020] Preferably, the volume concentration of the aqueous methanol solution is 40% to 60%, more preferably 50%. In the present application, the volume concentration of the aqueous methanol solution can be selected, for example, from 40%, 45%, 50%, 55%, 60%, and any point value within a range defined by any two of the above point values.

[0021] Preferably, the volume concentration of the aqueous phosphoric acid solution is 0.08% to 0.15%, preferably 0.1%. In the present application, the volume concentration of the aqueous phosphoric acid solution can be selected, for example, from 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, or 0.15%.

[0022] Preferably, the program of gradient elution comprises, in volume fraction of the mobile phase A and of the mobile phase B:

[0023] 0 min ~ 15 min, elution with 1 % ~ 5 % mobile phase A - 99 % ~ 95 % mobile phase B;

[0024] 15 min ~ 85 min, elution with 5 % ~ 10 % mobile phase A - 95 % ~ 90 % mobile phase B;

[0025] 85 min ~ 160 min, elution with 10 % ~ 26 % mobile phase A - 90 % ~ 74 % mobile phase B;

[0026] 160 min ~ 180 min, elution with 26 % ~ 50 % mobile phase A - 74 % ~ 50 % mobile phase B.

[0027] In a preferred embodiment of the application, the program of gradient elution comprises, in volume fraction of the mobile phase A and of the mobile phase B:

[0028] 0 min ~ 15 min, elution with 1 % mobile phase A - 99 % mobile phase B;

[0029] 15 min ~ 85 min, elution with 5 % mobile phase A - 95 % mobile phase B;

[0030] 85 min ~ 160 min, elution with 10 % mobile phase A - 90 % mobile phase B;

[0031] 160 min ~ 180 min, elution with 26 % mobile phase A - 74 % mobile phase B.

[0032] In another preferred embodiment of the application, the program of gradient elution comprises, in volume fraction of the mobile phase A and of the mobile phase B:

[0033] 0 min ~ 15 min, elution with 1 % mobile phase A - 99 % mobile phase B;

[0034] 15 min ~ 85 min, elution with 5 % mobile phase A - 95 % mobile phase B;

[0035] 85 min ~ 160 min, elution with 10 % mobile phase A - 90 % mobile phase B;

[0036] 160 min ~ 180 min, elution with 26 % mobile phase A - 74 % mobile phase B.

[0037] 180 min ~ 200 min, elution with 90 % mobile phase A - 10 % mobile phase B.

[0038] Preferably, the flow rate of the mobile phase is 0.8 mL / min ~ 1.2 mL / min, more preferably 1.0 mL / min. In the present application, the flow rate of the mobile phase can be selected, for example, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, etc.

[0039] Preferably, the column temperature of the chromatographic column is 25°C ~ 35°C, more preferably 30°C. For example, the column temperature of the chromatographic column can be selected 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C.

[0040] Preferably, the injection volume of the test sample solution and the control sample solution is 8 μL ~ 12 μL, more preferably 10 μL. For example, the injection volume of the test sample solution and the control sample solution can be selected 8 μL, 9 μL, 10 μL, 11 μL, or 12 μL.

[0041] In a second aspect, the present application provides a high performance liquid characteristic chromatogram obtained according to the above method for constructing a high performance liquid characteristic chromatogram and / or a fingerprint chromatogram of a traditional Chinese medicine composition for treating kidney disease.

[0042] Preferably, the above high performance liquid characteristic chromatogram comprises 4 characteristic peaks, and the retention time of each characteristic peak is within ±5% of the following specified values: 0.314 min (characteristic peak 1), 1.00 min (characteristic peak 2), and 1.042 min (characteristic peak 3), relative to the retention time of characteristic peak 4 (S) 1.124 ± 5% min.

[0043] Preferably, the peak area of each characteristic peak accounts for a percentage (%) of the total peak area within the following specified ranges: 4.00 ~ 8.00 (characteristic peak 1), 6.00 ~ 10.0 (characteristic peak 2), 35.0 ~ 10.0 (characteristic peak 3), and 1.00 ~ 2.50 (characteristic peak 4).

[0044] In a third aspect, the present application provides a high performance liquid fingerprint chromatogram obtained according to the above method for constructing a high performance liquid characteristic chromatogram and / or a fingerprint chromatogram of a traditional Chinese medicine composition for treating kidney disease.

[0045] Preferably, the above high performance liquid chromatography fingerprint comprises 21 fingerprint peaks, with fingerprint peak 14 as reference peak 14 (S), and the relative retention time of each fingerprint peak to S peak is calculated with the retention time of reference peak 14 (S) as 1.000 min; wherein the relative retention time of each fingerprint peak is within the range of ±5% of the following specified values: 0.020 min (fingerprint peak 1), 0.030 min (fingerprint peak 2), 0.183 min (fingerprint peak 3), 0.220 min (fingerprint peak 4), 0.314 min (fingerprint peak 5), 0.338 min (fingerprint peak 6), 0.371 min (fingerprint peak 7), 0.499 min (fingerprint peak 8), 0.589 min (fingerprint peak 9), 0.610 min (fingerprint peak 10), 0.645 min (fingerprint peak 11), 0.853 min (fingerprint peak 12), 0.860 min (fingerprint peak 13), 1.042 min (fingerprint peak 15), 1.068 min (fingerprint peak 16), 1.073 min (fingerprint peak 17), 1.098 min (fingerprint peak 18), 1.124 min (fingerprint peak 19), 1.145 min (fingerprint peak 20), and 1.231 min (fingerprint peak 21).

[0046] In a fourth aspect, the present application provides a method for detecting a traditional Chinese medicine composition for treating kidney disease, comprising: obtaining a high performance liquid chromatography spectrum of a test sample to be detected, and comparing the high performance liquid chromatography spectrum of the test sample with the high performance liquid characteristic spectrum of the traditional Chinese medicine composition for treating kidney disease, or comparing the high performance liquid chromatography spectrum of the test sample with the high performance liquid fingerprint spectrum of the traditional Chinese medicine composition for treating kidney disease.

[0047] In the present application, the similarity of the test sample spectrum and the fingerprint spectrum is calculated, and the similarity should not be less than 0.97.

[0048] In the present application, the total area of non-common peaks in the comparison of the test sample spectrum and the fingerprint spectrum should not be greater than 4% of the total peak area.

[0049] It should be noted that, in the process of constructing the characteristic spectrum or the fingerprint spectrum, a standby solution prepared from a known traditional Chinese medicine composition for treating kidney disease, such as Xueyuanan capsules or tablets, at a specific concentration is referred to as a "test sample solution". When the characteristic spectrum or the fingerprint spectrum is used to detect or identify whether an unknown substance is the traditional Chinese medicine composition for treating kidney disease of the present application, a solution prepared from the unknown substance to be detected or identified is referred to as a "test sample solution".

[0050] The present application has at least the following beneficial effects:

[0051] (1) The present application provides a traditional Chinese medicine composition for treating kidney disease, in particular, a high performance liquid characteristic map and a fingerprint map of Xue Nian Capsules or tablets and a construction method thereof, which effectively constructs the characteristic map and the fingerprint map through high performance liquid chromatography, and provides a high performance and reliable guarantee for more reliable and more accurate analysis, detection and identification of the traditional Chinese medicine composition for treating kidney disease.

[0052] (2) The construction method of the high performance liquid characteristic map and the fingerprint map of the traditional Chinese medicine composition for treating kidney disease provided by the present application has good stability and repeatability for single product and multiple batches, and good peak separation and stable peak shape, and has good reliability and high accuracy for identification and detection of the traditional Chinese medicine composition for treating kidney disease. BRIEF DESCRIPTION OF DRAWINGS

[0053] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0054] Figure 1 HPLC chromatogram results of the solvent of the blank experiment sample in Example 3;

[0055] Figure 2 HPLC chromatogram results of the precision investigation in Example 3;

[0056] Figure 3 HPLC chromatogram results of the stability investigation in Example 3;

[0057] Figure 4 HPLC chromatogram results of the repeatability investigation in Example 3;

[0058] Figure 5 HPLC map and common mode map results of 10 batches of Xue Nian Capsules in Example 4;

[0059] Figure 6 HPLC fingerprint map results determined in Example 4;

[0060] Figure 7 Schematic map for determining the HPLC characteristic peak map in Example 5;

[0061] Figure 8 HPLC characteristic map results determined in Example 5. DETAILED DESCRIPTION

[0062] The present application will be further described in detail below with reference to the specific embodiments, and the embodiments are given only to illustrate the present application, but not to limit the scope of the present application.

[0063] The information of reagents and reagents, instruments used in the embodiments of the present application is as follows.

[0064] Reagents and reagents: reference substance berberine hydrochloride (batch number 110713-202015, purity 85.9%), rosmarinic acid (111871-202007, purity 98.1%), chlorogenic acid (110753-202018, purity 96.1%), buddleoside (111528-201911, purity 98.5%), ursolic acid (110742-201823, purity 99.9%), phellodendrine hydrochloride (111895-201805, purity 94.9%) were purchased from China Institute for Drug Control; acetonitrile was chromatographically pure, water was ultrapure water, and the rest of the reagents were analytical pure. Xue Nian'an capsule samples (produced by Yunnan Lei Yunshang Ideal Pharmaceutical Co., Ltd.) a total of 10 batches, batch number as shown in Table 1.

[0065]

[0066] Instrument: Agilent 1260 analytical high performance liquid chromatograph; balance: OHAUS AR224-N electronic balance (one ten thousandth), EX125ZH electronic analytical balance (one hundred thousandth), Ohaus Instrument (Changzhou) Co., Ltd.; ultrasonic instrument: SB25-12DTD ultrasonic instrument, Ningbo Xinzhi Biological Technology Co., Ltd.; Sartius PB-21 type pH meter.

[0067] Example 1 Preparation of solution

[0068] Preparation of test solution

[0069] The Xue Nian'an capsule was removed from the shell, the contents were mixed, 1.5 g was accurately weighed in a stoppered triangular flask, 50% methanol solution 50 mL was added, the weight was determined, ultrasonic extraction was carried out for 30 min (power 500 W, frequency 40 kHz), it was taken out and cooled, 50% methanol was added to make up the weight, filtered, the filtrate was passed through a 0.45 μm microporous filter membrane, the filtrate was taken, and the test solution was obtained for standby.

[0070] Preparation of reference solution

[0071] An appropriate amount of rosmarinic acid reference substance was accurately weighed and added to methanol to prepare a solution containing 20 μg per 1 mL, and a rosmarinic acid reference solution was obtained for standby.

[0072] Accurately weigh 10 mg each of berberine hydrochloride, rosmarinic acid, chlorogenic acid, monardin, ursolic acid, and phellodendronine hydrochloride and place them in 50 mL volumetric flasks. Dissolve each in methanol and dilute to the mark to serve as reference stock solutions. Measure 5 mL of each of the six reference stock solutions in a 50 mL volumetric flask, add methanol to the mark, shake well, filter through a 0.45 μm microporous membrane, and collect the filtrate to obtain a mixed reference solution for later use.

[0073] Example 2 High Performance Liquid Chromatography Injection

[0074] Chromatographic conditions or system suitability test (determination according to the high-performance liquid chromatography method (General Chapter 0512) in the Chinese Pharmacopoeia (2020 edition)): octadecylsilane bonded silica gel (Agilent ZORBAX SB-C18, 4.6 mm × 250 mm, 5 μm) was used as the packing material; acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid aqueous solution was used as the mobile phase B, following the elution gradient specified in Table 2; the flow rate was 1.0 mL / min. The detection wavelength was 330 nm. The column temperature was 30°C. The number of theoretical plates calculated based on rosmarinic acid should be no less than 400,000.

[0075]

[0076] Accurately pipette 10 µL of rosmarinic acid reference solution and test solution respectively, inject them into liquid chromatograph, measure, and record the chromatogram within at least 160 min, for example, the chromatogram can be recorded within 180 min.

[0077] Example 3: Injection performance investigation

[0078] Blank test

[0079] In order to confirm whether the sample solution would interfere with the chromatographic results, as a blank experiment, 10 μL of 50% methanol aqueous solution (i.e., sample solvent) was accurately aspirated and injected into the HPLC for measurement under the same injection conditions as in Example 2. The results are shown in Figure 2. Figure 1 As shown, it shows that the sample solvent has no interference with the determination results.

[0080] Determination of determination time

[0081] The test solution was injected under the same injection conditions as in Example 2. It was found that there was no chromatographic peak after the retention time of 180 min. Therefore, the measurement time was determined to be before 180 min.

[0082] Precision investigation

[0083] The same sample solution was precisely taken 10 μL, and 6 injections were performed successively under the same injection conditions as in Example 2. The chromatograms and the common pattern chromatogram R are shown in Figure 2 .

[0084] The obtained chromatograms were introduced into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) developed and established by the National Medical Products Administration, and the similarity was calculated by taking rosmarinic acid (B) as the reference peak. The common pattern chromatogram R was generated by using the "median method". Compared with the common pattern chromatogram R, the similarity of the chromatograms of the 6 injections was 1.000 (the similarity should be above 0.90), indicating that the precision of the instrument was good and met the requirements.

[0085] Stability investigation

[0086] The same sample solution was precisely taken 10 μL, and 6 injections were performed successively under the same injection conditions as in Example 2. The chromatograms and the common pattern chromatogram R are shown in Figure 3 .

[0087] The obtained chromatograms were introduced into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) developed and established by the National Medical Products Administration, and the similarity was calculated by taking rosmarinic acid (B) as the reference peak. The common pattern chromatogram R was generated by using the "median method". Compared with the common pattern chromatogram R, the similarity of the chromatograms of the 6 injections was 1.000 (the similarity should be above 0.90), indicating that the precision of the instrument was good and met the requirements.

[0088] Reproducibility investigation

[0089] The same sample solution was precisely taken 10 μL, and 6 injections were performed successively under the same injection conditions as in Example 2. The chromatograms and the common pattern chromatogram R are shown in Figure 4 .

[0090] The obtained chromatograms were introduced into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) developed and established by the National Medical Products Administration, and the similarity was calculated by taking rosmarinic acid (B) as the reference peak. The common pattern chromatogram R was generated by using the "median method". Compared with the common pattern chromatogram R, the similarity of the chromatograms of the 6 injections was 1.000 (the similarity should be above 0.90), indicating that the precision of the instrument was good and met the requirements.

[0091] Example 4 Fingerprint

[0092] The control sample solution and the prepared test sample solution of 10 batches of Xuean Capsules were injected under the same injection conditions as in Example 2, and the chromatograms were recorded.

[0093] The HPLC chromatogram results were introduced into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version). Since peak 14 (rosmarinic acid) had good peak separation, a large peak, and stable peak shape, and was a common peak, it was used as a reference peak for retention time calibration and matching, similarity calculation, and generation of a common mode spectrum R using the "median method". The HPLC fingerprints of 10 batches of drugs (sample numbers S1-S10) and the common mode are shown in Figure 5 , and the similarity calculation results are shown in Table 3.

[0094]

[0095] By analyzing and comparing the obtained spectrum results, 21 common peaks (peaks 1-21) with a total peak area of more than 1% were calibrated, as shown in Figure 6 . After comparison with the control sample, 4 of the peaks were chlorogenic acid (peak 5), rosmarinic acid (peak 14), berberine hydrochloride (peak 15), and buddlejaflavone (peak 19).

[0096] Taking peak 14 as the S peak, the relative retention time (the ratio of the retention time of each peak to that of rosmarinic acid) and the relative peak area (the ratio of the peak area of each peak to that of rosmarinic acid) of each fingerprint peak to the S peak were calculated. The relative retention time should be within ±5% of the specified value. The relative retention time specified value is summarized in Table 4, and the relative peak area value is summarized in Table 5.

[0097]

[0098] From the above results, it can be found that the RSD of the relative retention time of the 21 fingerprint peaks is 0.04% to 1.01%, with high positioning accuracy, indicating that the relative retention time can be used to position each fingerprint peak. The RSD of the relative peak area is 9.30% to 38.25%, with a slight variation, but the peak area variation coefficients of most peaks (19 peaks) are below 30%, and two peaks (peaks 11 and 21) are 32.19% and 38.25%.

[0099] Similarity evaluation method and limit standard

[0100] The HPLC chromatograms of each batch of capsules and the control fingerprint DZ were introduced into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) for pairwise comparison. Taking rosmarinic acid (peak 14) as the reference peak B, the retention time was calibrated and matched, and the similarity of the HPLC chromatogram of the drug sample to the control fingerprint DZ was calculated.

[0101] From the similarity calculation results of Table 3 and Table 6, it can be seen that the similarity of 10 batches of drugs together compared with DZ spectrum (Table 3) and each drug sample compared with DZ spectrum (Table 6) is different, but the difference is not big. The similarity of the former is 0.994~0.999; the similarity of the latter is 0.971~1.00. Overall, the established fingerprint spectrum DZ has good stability and reproducibility whether it is compared with batch samples or single sample, and it is suggested that the similarity of 0.97 should be used as the internal control limit standard for judging the quality stability of different batches of drugs.

[0102]

[0103] Fingerprint peak relative retention time evaluation

[0104] According to the provisions of the 2020 edition of Chinese Pharmacopoeia for the evaluation of traditional Chinese medicine injection fingerprint spectrum, the HPLC fingerprint peak area ratio is used for fingerprint spectrum evaluation, and the requirements for peak area ratio are as follows:

[0105] (1) The common peaks with retention time less than or equal to 30 min, Ai (single peak area) ≥20% At (total peak area), RSD≤ ±20%; 10% At≤Ai<20% At, RSD≤ ±25%; 5% At≤Ai<10% At, RSD≤ ±30%; Ai<5% At, the peak area ratio is not required, but the relative retention time must be calibrated; (2) The common peaks with retention time more than 30 min, Ai≥10% At, as above; Ai<10% At, the peak area ratio is not required, but the relative retention time must be calibrated.

[0106] The relative retention time and relative peak area of 21 fingerprint peaks in the HPLC fingerprint spectrum of 10 batches of Xue Nian'an capsules are shown in Table 4 and Table 5, among which the retention time of peaks 1~4 is 2.77~29.58 min, and the rest is after 41.83 min (peak 5); among them, peaks 5, 9, 14, and 15 account for more than 5% of the total peak area, peak 9 accounts for more than 10% of the total peak area, and peak 15 accounts for more than 20% of the total peak area, and this peak is also the reference peak. The drug standard has a content determination item, so the evaluation of common fingerprint peaks of Xue Nian'an capsules mainly needs to calibrate 2 common fingerprint peaks.

[0107] According to the determination results of the fingerprint spectrum of 10 batches of drugs, peak 14 (rosmarinic acid) is used as the reference peak to calculate the relative retention time (RRt, the ratio of single peak to rosmarinic acid peak retention time) of 21 fingerprints in the standard. The HPLC spectrum of the standard should be as follows: Figure 6The RRt values of each peak should be within ± 5% of the values specified for each corresponding peak number in Table 7.

[0108]

[0109] Analysis of the proportion of total peak area of common peaks and non-common peaks

[0110] The HPLC chromatograms of each batch of capsules and the control fingerprint DZ were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) for pairwise comparison. With peak 14 (rosmarinic acid) as the reference peak, the retention time was calibrated and matched, and by comparing with the control fingerprint DZ, the common peaks, non-common peaks and 21 fingerprint peaks of the sample HPLC chromatogram and the control fingerprint were determined, and the total peak area percentage of the HPLC fingerprint chromatogram was calculated, as shown in Table 8.

[0111] The total peak area ratio of the common peaks of 10 batches of samples to the total peak area of the HPLC chromatogram was 96.2%~98.3%, with an average of 97.3% and an RSD of 0.65%, indicating that the control fingerprint DZ represented the overall characteristics of the chemical components of Xue Nian An capsules, with good stability and could be used for drug quality characterization. The total peak area ratio of the 21 common fingerprint peaks was 88.3%~94.6%, with an average of 92.0% and an RSD of 2.10%, indicating that the 21 fingerprint peaks could better reflect the overall characteristics of the HPLC fingerprint chromatogram, and the values were relatively stable, also indicating that the quality of Xue Nian An capsules of different batches was stable.

[0112] The total peak area ratio of the non-common peaks to the total peak area was compared with the control fingerprint DZ. The non-common peaks appearing in the sample HPLC chromatogram were peaks that were not in the DZ chromatogram and were added in the sample. The total peak area variation of the non-common peaks of different batches of samples was large, ranging from 1.7% to 3.8%, with an average of 2.7% and an RSD of 23.2%.

[0113] The variation of the non-common peak area ratio is often related to the source and quality of medicinal materials and needs to be closely monitored. According to the requirements of the 2020 edition of "Chinese Pharmacopoeia" for traditional Chinese medicine injections, the total area of non-common peaks should not be greater than 5% of the total peak area when comparing the standard or test sample chromatogram with the fingerprint chromatogram. According to the results of this experiment, it is recommended that the total area of non-common peaks should not be greater than 4% of the total peak area as the internal control limit standard for the stability of Xue Nian An capsules, that is, when comparing the standard or test sample chromatogram with the fingerprint chromatogram, the total area of non-common peaks, whether added or missing, should not be greater than 4% of the total peak area.

[0114]

[0115] In summary, according to the similarity evaluation system of Chinese medicine chromatographic fingerprint, (1) the similarity between the standard or test sample spectrum and the reference fingerprint spectrum shall not be less than 0.97; (2) there shall be 21 fingerprint peaks in the standard or test sample spectrum, and the peak 14 corresponding to the reference is the S peak. The relative retention time of each fingerprint peak and the S peak shall be calculated, and the relative retention time shall be within ±5% of the specified value; (3) the total area of ​​non-common peaks shall not be greater than 4% of the total peak area when the standard or test sample spectrum is compared with the fingerprint spectrum. The specified values ​​of the relative retention time of each fingerprint peak in the fingerprint spectrum are shown in Table 9. Figure 6 Integration parameters: slope sensitivity of 1.0, peak width of 0.02, minimum peak area of ​​1, and minimum peak height of 1.7.

[0116]

[0117] Example 5 Characteristic Spectrum

[0118] From the percentage data of each fingerprint peak in the total peak area in Table 5 above, it can be found that peak 15 (berberine) has the highest ratio of the total peak area; peak 5 (chlorogenic acid), peak 9 and peak 14 (rosmarinic acid) are second; the peak area ratios of the remaining 17 peaks are all smaller than that of peak 5.

[0119] Since the peak areas of most fingerprint peaks are very small, four fingerprint peaks with clear attribution, RSD ≤ 30% and belonging to different medicinal materials are selected as characteristic peaks. Figure 7 As shown, with peak 14 (rosmarinic acid) as the reference peak, the relative retention times of the remaining three peaks (the ratio of the retention time of the single peak to the reference peak) were calculated for the location of the characteristic peaks. For four of the peaks (peaks 5, 14, 15, and 19), the average of the ratio of each peak to the total peak area was adjusted and relaxed by 20% (±20%) based on the average of the ratio of each peak to the total peak area. The limits were adjusted and relaxed based on the results of 10 batches of samples, and a reference standard for the limit of the ratio of each peak to the total peak area was established (Table 10). Peak 9 was only located. The chromatogram of the standard or test sample was compared with the characteristic chromatogram. The order of the four characteristic peaks is as follows: Figure 7 As shown in Table 10, the relative retention time should comply with the requirements of Table 10, and the percentage of the four characteristic peaks to the total peak area should be within the range specified in Table 10.

[0120]

[0121] In summary, there should be 4 characteristic peaks in the characteristic map, and the peak 4 corresponding to the reference substance is S peak. The relative retention time of each characteristic peak to the S peak should be within ±5% of the specified value, and the specified value is: 0.314 (peak 1, chlorogenic acid), 1.00 (peak 2, rosmarinic acid), 1.042 (peak 3, berberine), and 1.124 (peak 4, geniposide). The percentage of the peak area of peaks 1-4 to the total peak area should be within the specified range, and the specified range is (%): 4.00-8.00 (peak 1), 6.00-10.0 (peak 2), 35.0-10.0 (peak 3), and 1.00-2.50 (peak 4). The characteristic map is shown in Figure 8 The integral parameters are: the slope sensitivity is 1.0, the peak width is 0.02, the minimum peak area is 1, and the minimum peak height is 1.7.

[0122] The above is only a few exemplary embodiments of the present application, and does not limit the present application in any form. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any skilled person in the art can make some changes or modifications to the above disclosed technical contents without departing from the scope of the technical solutions of the present application, and such changes or modifications are equivalent to equivalent embodiments, and are within the scope of the technical solutions of the present application.

Claims

1. A method for constructing a fingerprint of a traditional Chinese medicine composition for treating kidney disease, comprising: (a) preparing a test solution of the traditional Chinese medicine composition for treating kidney disease and a control solution; wherein the traditional Chinese medicine composition for treating kidney disease comprises 36-44 parts by weight of Orthosiphon aristatus, 54-66 parts by weight of Herba Hedyseidis Rubrae, 72-88 parts by weight of Rhizoma Imperatae, and 10-14 parts by weight of Cortex Phellodendri; and the control comprises berberine, rosmarinic acid, chlorogenic acid, geniposide, ursolic acid, and phellodendrine hydrochloride; (b) performing high performance liquid chromatography on the test solution and the control solution; wherein the high performance liquid chromatography comprises: using octadecylsilane-bonded silica gel as the filler of a chromatographic column, using acetonitrile as mobile phase A and using phosphoric acid aqueous solution as mobile phase B, performing gradient elution, the theoretical plate number calculated based on rosmarinic acid being not less than 400000, and the gradient elution program comprising: 0 min-15 min, elution with 1%-5% mobile phase A-99%-95% mobile phase B; 15 min-85 min, elution with 5%-10% mobile phase A-95%-90% mobile phase B; 85 min-160 min, elution with 10%-26% mobile phase A-90%-74% mobile phase B; 160 min-180 min, elution with 26%-50% mobile phase A-74%-50% mobile phase B.

2. The method of claim 1, wherein, The traditional Chinese medicine composition for treating kidney disease comprises 40 parts by weight of Orthosiphon aristatus, 60 parts by weight of Herba Hedyseidis Rubrae, 80 parts by weight of Rhizoma Imperatae, and 12 parts by weight of Cortex Phellodendri.

3. The method of claim 1, wherein, In step (a), the method for preparing the test solution comprises: dissolving a sample of the traditional Chinese medicine composition for treating kidney disease in a methanol aqueous solution, performing ultrasonic extraction, filtering, and quantifying to obtain the test solution.

4. The method of claim 1, wherein, The concentration of the test solution is 10 mg / mL-40 mg / mL.

5. The method of claim 4, wherein, The concentration of the test solution is 30 mg / mL.

6. The method of claim 1, wherein, The concentration of the control solution is 10 μg / mL-30 μg / mL.

7. The method of claim 6, wherein, The concentration of the control solution is 20 μg / mL.

8. The method of claim 3, wherein, The ultrasonic extraction is performed at a power of 400 W-600 W and a frequency of 30 kHz-50 kHz for 20 min-40 min.

9. The method of claim 3, wherein, The methanol aqueous solution has a volume concentration of 40%-60%.

10. The method of claim 9, wherein, The methanol aqueous solution has a volume concentration of 50%.

11. The method of claim 1, wherein, The phosphoric acid aqueous solution has a volume concentration of 0.08%-0.15%.

12. The method of claim 11, wherein, The phosphoric acid aqueous solution has a volume concentration of 0.10%.

13. The method of claim 1, wherein, The flow rate of the mobile phase is 0.8 mL / min-1.2 mL / min, and the column temperature of the chromatographic column is 25°C-35°C.

14. The method of claim 13, wherein, The flow rate of the mobile phase is 1.0 mL / min, and the column temperature of the chromatographic column is 30°C.

15. The method of claim 1, wherein, The injection volume of the test solution and the control solution is 8 μL-12 μL.

16. The method of claim 15, wherein, The injection volume of the test solution and the control solution is 10 μL.

17. The method of any one of claims 1 to 16, wherein, The constructed fingerprint of the traditional Chinese medicine composition for treating kidney disease contains four characteristic peaks, and the retention time of the characteristic peak of geniposide is 1.124±5% min, and the retention times of the other three characteristic peaks are within ±5% of the following specified values: the characteristic peak of chlorogenic acid is 0.314 min, the characteristic peak of rosmarinic acid is 1.00 min, and the characteristic peak of berberine hydrochloride is 1.042 min.

18. The method of claim 17, wherein, The peak area of the four characteristic peaks accounts for 4.00-8.00%, 6.00-10.0%, 35.0-10.0%, and 1.00-2.50% of the total peak area, respectively.

19. The method of any one of claims 1 to 16, wherein, The constructed fingerprint of the traditional Chinese medicine composition for treating kidney disease contains 21 fingerprint peaks, and the relative retention times of the other 20 fingerprint peaks are calculated with the retention time of the characteristic peak of rosmarinic acid as the reference, and are within ±5% of the following specified values: 0.020 min, 0.030 min, 0.183 min, 0.220 min, 0.314 min, 0.338 min, 0.371 min, 0.499 min, 0.589 min, 0.610 min, 0.645 min, 0.853 min, 0.860 min, 1.042 min, 1.068 min, 1.073 min, 1.098 min, 1.124 min, 1.145 min, and 1.231 min.

20. A method for detecting a traditional Chinese medicine composition for treating kidney disease, comprising: constructing a fingerprint of the traditional Chinese medicine composition for treating kidney disease according to the method of any one of claims 1 to 19; obtaining a high-performance liquid chromatogram of a test sample to be detected; and comparing the high-performance liquid chromatogram of the test sample with the constructed fingerprint.

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