Establishment of HPLC characteristic chromatogram of Ejiao Qianggu oral liquid

Abstract

The present application relates to the technical field of traditional Chinese medicine quality detection, and is a construction method of HPLC characteristic spectrum of Ejiao Qianggu oral liquid, which is performed according to the following steps: preparing test sample solution and mixed control solution respectively, sucking the test sample solution and the mixed control solution into a high performance liquid chromatograph, and obtaining the HPLC characteristic spectrum of Ejiao Qianggu oral liquid.The HPLC characteristic spectrum obtained by the construction method of HPLC characteristic spectrum of Ejiao Qianggu oral liquid has good peak shape, high separation degree, high similarity, strong specificity, good stability, and is accurate and reliable; the internal quality of Ejiao Qianggu oral liquid can be controlled and evaluated comprehensively, and the method can be used for drug quality standard and quality control of Ejiao Qianggu oral liquid.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine quality detection, and is a method for constructing an HPLC characteristic spectrum of donkey-hide gelatin bone-strengthening oral liquid. Background Art

[0002] Donkey-hide gelatin bone strengthening oral liquid is composed of six medicinal ingredients: Rehmannia root, Astragalus root, Codonopsis pilosula, Lycium barbarum, donkey-hide gelatin, and oyster shell. The standard is included in the National Drug Standards, numbered WS3-155(Z-017)-2008Z, which discloses the quality control methods for donkey-hide gelatin bone strengthening oral liquid. Due to the lack of specificity in the current standard, it is difficult to fully control the intrinsic quality of the drug preparation and ensure its safety and effectiveness. Summary of the Invention

[0003] The present invention provides a method for constructing an HPLC characteristic spectrum of donkey-hide gelatin bone-strengthening oral liquid, which overcomes the shortcomings of the above-mentioned prior art and can effectively solve the problem that the current standards are difficult to fully control the intrinsic quality of donkey-hide gelatin bone-strengthening oral liquid.

[0004] The technical solution of the present invention is achieved by the following measures: a method for constructing an HPLC characteristic spectrum of donkey-hide gelatin bone-strengthening oral liquid is carried out according to the following steps:

[0005] Preparation of test solution: Accurately measure donkey-hide gelatin bone strengthening oral liquid, add anhydrous ethanol to mix, sonicate, filter, concentrate the filtrate to near dryness, add purified water to dissolve, extract with petroleum ether, discard the upper petroleum ether liquid, extract the lower water liquid with ethyl acetate, collect the ethyl acetate extract and concentrate it to near dryness, dissolve it with methanol, shake well, filter, and obtain the test solution;

[0006] Preparation of mixed reference solution: Accurately weigh dangshenin, calycosin, calycosin, formononetin and formononetin reference substances, respectively, and dissolve them in methanol to obtain a mixed reference solution;

[0007] Construction of characteristic spectrum: Accurately aspirate the mixed reference solution and test solution respectively, inject them into high performance liquid chromatography to obtain the HPLC characteristic spectrum of donkey-hide gelatin bone strengthening oral liquid.

[0008] The following are further optimizations and / or improvements to the above technical solutions:

[0009] In the chromatographic conditions of the above-mentioned high performance liquid chromatograph, the chromatographic column is a C18 chromatographic column, 250 mm×4.6 mm, 5 μm.

[0010] In the chromatographic conditions of the above-mentioned high performance liquid chromatograph, the mobile phase is an acetonitrile-phosphoric acid system, in which phase A is acetonitrile and phase B is a phosphoric acid solution with a mass percentage concentration of 0.05% to 0.15%. Gradient elution is adopted, and the elution gradient includes:

[0011] From 0 min to 20 min, phase A increased from 10% to 25%, and phase B decreased from 90% to 75%;

[0012] From 20 to 40 minutes, phase A increased from 25% to 35%, and phase B decreased from 75% to 65%;

[0013] From 40 to 60 minutes, phase A increased from 35% to 50%, and phase B decreased from 65% to 50%.

[0014] The chromatographic conditions of the above-mentioned high performance liquid chromatograph were as follows: a mobile phase flow rate of 1.0 mL / min, a column temperature of 30° C., an ultraviolet detector, and a detection wavelength of 267 nm.

[0015] In the preparation of the above-mentioned test solutions, the volume ratio of donkey-hide gelatin bone strengthening oral solution to anhydrous ethanol is 1:4 to 6, the volume ratio of donkey-hide gelatin bone strengthening oral solution to purified water is 1:1 to 3, and the volume ratio of donkey-hide gelatin bone strengthening oral solution to methanol is 1:5.

[0016] The concentrations of the reference substances in the mixed reference solution were 1.81 mg / mL of codonopsis glycoside, 4.87 mg / mL of calycosin, 7.66 mg / mL of calycosin, 4.23 mg / mL of formononetin, and 6.71 mg / mL of formononetin.

[0017] In the preparation of the above test solution, the ultrasonic time is 1.0h to 1.5h.

[0018] The beneficial effects of the present invention are:

[0019] The present invention uses HPLC technology to establish a characteristic spectrum of the compound system of donkey-hide gelatin bone strengthening oral liquid, identifies five common peaks, and can comprehensively control and evaluate the intrinsic quality of donkey-hide gelatin bone strengthening oral liquid, and is used for the pharmaceutical quality standard and quality control of donkey-hide gelatin bone strengthening oral liquid;

[0020] The present invention has the advantages of low organic solvent usage, good purification and concentration effects, simple and quick method, high sensitivity and easy operation, etc.

[0021] The HPLC characteristic spectrum of the donkey-hide gelatin bone-strengthening oral liquid obtained by the present invention has good peak shape, high separation, easy identification, high similarity, strong specificity, good stability, accuracy and reliability.

[0022] In summary, the HPLC characteristic spectrum obtained by the method for constructing the HPLC characteristic spectrum of the donkey-hide gelatin bone-strengthening oral liquid of the present invention has good peak shape, high separation, easy identification, high similarity, strong specificity, good stability, accuracy and reliability; it can more comprehensively control and evaluate the intrinsic quality of the donkey-hide gelatin bone-strengthening oral liquid, and be used for the pharmaceutical quality standard and quality control of the donkey-hide gelatin bone-strengthening oral liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 HPLC chromatogram comparison of the mixed reference solution and the test solution prepared in Example 11 of the present invention;

[0024] Figure 2 HPLC chromatogram comparison of the Astragalus membranaceus negative sample solution and the Codonopsis pilosula negative sample solution prepared in Example 11 of the present invention;

[0025] Figure 3 This is an overlay of HPLC characteristic spectra of 15 batches of donkey-hide gelatin bone-strengthening oral liquid described in Example 11 of the present invention;

[0026] in, Figure 1 A is the HPLC chromatogram of the mixed reference solution, and B is the HPLC chromatogram of the test solution; Figure 2 C is the HPLC chromatogram of the negative sample solution of Astragalus membranaceus, and D is the HPLC chromatogram of the negative sample solution of Codonopsis pilosula; Figure 3 Peak 1 is calycosin, peak 2 is codonopsis pilosula, peak 3 is formononetin, peak 4 is calycosin and peak 5 is formononetin. DETAILED DESCRIPTION

[0027] The present invention is not limited to the following embodiments, and specific implementation methods can be determined based on the technical solutions of the present invention and actual conditions. Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all chemical reagents and chemicals commonly known in the prior art; unless otherwise specified, percentages in the present invention are all percentages by mass; unless otherwise specified, solutions in the present invention are all aqueous solutions with water as the solvent, for example, a hydrochloric acid solution is an aqueous hydrochloric acid solution.

[0028] The present invention will be further described below in conjunction with the embodiments:

[0029] Example 1: The method for constructing the HPLC characteristic spectrum of the donkey-hide gelatin bone-strengthening oral liquid is carried out according to the following steps:

[0030] Preparation of test solution: Accurately measure donkey-hide gelatin bone strengthening oral liquid, add anhydrous ethanol to mix, sonicate, filter, concentrate the filtrate to near dryness, add purified water to dissolve, extract with petroleum ether, discard the upper petroleum ether liquid, extract the lower water liquid with ethyl acetate, collect the ethyl acetate extract and concentrate it to near dryness, dissolve it with methanol, shake well, filter, and obtain the test solution;

[0031] Preparation of mixed reference solution: Accurately weigh dangshenin, calycosin, calycosin, formononetin and formononetin reference substances, respectively, and dissolve them in methanol to obtain a mixed reference solution;

[0032] Construction of characteristic spectrum: Accurately aspirate the mixed reference solution and test solution respectively, inject them into high performance liquid chromatography to obtain the HPLC characteristic spectrum of donkey-hide gelatin bone strengthening oral liquid.

[0033] Example 2: As an optimization of the above example, in the chromatographic conditions of the high performance liquid chromatograph, the chromatographic column is a C18 chromatographic column, 250 mm×4.6 mm, 5 μm.

[0034] Example 3: As an optimization of the above example, the chromatographic conditions of the high performance liquid chromatograph are as follows: the mobile phase is an acetonitrile-phosphoric acid system, the A phase of the acetonitrile-phosphoric acid system is acetonitrile, and the B phase is a phosphoric acid solution with a mass percentage concentration of 0.05% to 0.15%. Gradient elution is adopted, and the elution gradient includes:

[0035] From 0 min to 20 min, phase A increased from 10% to 25%, and phase B decreased from 90% to 75%;

[0036] From 20 to 40 minutes, phase A increased from 25% to 35%, and phase B decreased from 75% to 65%;

[0037] From 40 to 60 minutes, phase A increased from 35% to 50%, and phase B decreased from 65% to 50%.

[0038] Example 4: As an optimization of the above example, the chromatographic conditions of the high performance liquid chromatograph are as follows: the mobile phase flow rate is 1.0 mL / min, the column temperature is 30° C., the detector is an ultraviolet detector, and the detection wavelength is 267 nm.

[0039] Example 5: As an optimization of the above examples, in the preparation of the test solution, the volume ratio of donkey-hide gelatin bone strengthening oral solution to anhydrous ethanol is 1:4 to 6, the volume ratio of donkey-hide gelatin bone strengthening oral solution to purified water is 1:1 to 3, and the volume ratio of donkey-hide gelatin bone strengthening oral solution to methanol is 1:5.

[0040] Example 6: As an optimization of the above example, the concentrations of the reference substances in the mixed reference solution were 1.81 mg / mL of dangshenylin, 4.87 mg / mL of calycosin, 7.66 mg / mL of calycosin, 4.23 mg / mL of formononetin, and 6.71 mg / mL of formononetin.

[0041] Example 7: As an optimization of the above example, in the preparation of the test solution, the ultrasonic time is 1.0h to 1.5h.

[0042] Example 8:

[0043] The method for constructing the HPLC characteristic spectrum of the donkey-hide gelatin bone-strengthening oral liquid is carried out according to the following steps:

[0044] Preparation of the test solution: accurately measure 10 mL of donkey-hide gelatin bone strengthening oral liquid, add 40 mL of anhydrous ethanol and vortex until thoroughly mixed, sonicate for 1.0 h, filter, evaporate the filtrate in a water bath until nearly dry, add 10 mL of purified water to dissolve, extract with petroleum ether, discard the upper petroleum ether liquid, extract the lower water liquid with ethyl acetate, collect the ethyl acetate extract, evaporate to dryness in a water bath, dilute to 50 mL with methanol, shake well, and filter through 0.45 μm to obtain the test solution;

[0045] Preparation of mixed reference solution: Accurately weigh dangshenin, calycosin, calycosin, formononetin, and formononetin reference substance, and dilute to 50 mL with methanol to obtain a mixed reference solution containing 1.81 mg / mL dangshenin, 4.87 mg / mL calycosin, 7.66 mg / mL calycosin, 4.23 mg / mL formononetin, and 6.71 mg / mL formononetin.

[0046] Construction of characteristic spectrum: Accurately aspirate 10 μL of the mixed reference solution and the test solution respectively, inject them into the high performance liquid chromatograph to obtain the HPLC characteristic spectrum of the donkey-hide gelatin bone strengthening oral liquid.

[0047] Among them, the chromatographic conditions of the high performance liquid chromatography are:

[0048] The chromatographic column was an InertSustainAQ-C18 column, 250 mm × 4.6 mm, 5 μm;

[0049] The mobile phase is an acetonitrile-phosphoric acid system, in which phase A is acetonitrile and phase B is a 0.05% by mass phosphoric acid solution. Gradient elution is used, and the elution gradient includes:

[0050] From 0 min to 20 min, phase A increased from 10% to 25%, and phase B decreased from 90% to 75%;

[0051] From 20 to 40 minutes, phase A increased from 25% to 35%, and phase B decreased from 75% to 65%;

[0052] From 40 to 60 minutes, phase A increased from 35% to 50%, and phase B decreased from 65% to 50%.

[0053] The mobile phase flow rate was 1.0 mL / min, the column temperature was 30°C, the detector was an ultraviolet detector, and the detection wavelength was 267 nm.

[0054] Example 9:

[0055] The method for constructing the HPLC characteristic spectrum of the donkey-hide gelatin bone-strengthening oral liquid is carried out according to the following steps:

[0056] Preparation of the test solution: accurately measure 10 mL of donkey-hide gelatin bone strengthening oral liquid, add 60 mL of anhydrous ethanol and vortex until thoroughly mixed, sonicate for 1.5 h, filter, evaporate the filtrate in a water bath until nearly dry, add 30 mL of purified water to dissolve, extract with petroleum ether, discard the upper petroleum ether liquid, extract the lower water liquid with ethyl acetate, collect the ethyl acetate extract, evaporate to dryness in a water bath, dilute to 50 mL with methanol, shake well, and filter through 0.45 μm to obtain the test solution;

[0057] Preparation of mixed reference solution: Accurately weigh dangshenin, calycosin, calycosin, formononetin, and formononetin reference substance, and dilute to 50 mL with methanol to obtain a mixed reference solution containing 1.81 mg / mL dangshenin, 4.87 mg / mL calycosin, 7.66 mg / mL calycosin, 4.23 mg / mL formononetin, and 6.71 mg / mL formononetin.

[0058] Construction of characteristic spectrum: Accurately aspirate 10 μL of the mixed reference solution and the test solution respectively, inject them into the high performance liquid chromatograph to obtain the HPLC characteristic spectrum of the donkey-hide gelatin bone strengthening oral liquid.

[0059] Among them, the chromatographic conditions of the high performance liquid chromatography are:

[0060] The chromatographic column was an InertSustainAQ-C18 column, 250 mm × 4.6 mm, 5 μm;

[0061] The mobile phase is an acetonitrile-phosphoric acid system, in which phase A is acetonitrile and phase B is a 0.15% by mass phosphoric acid solution. Gradient elution is used, and the elution gradient includes:

[0062] From 0 min to 20 min, phase A increased from 10% to 25%, and phase B decreased from 90% to 75%;

[0063] From 20 to 40 minutes, phase A increased from 25% to 35%, and phase B decreased from 75% to 65%;

[0064] From 40 to 60 minutes, phase A increased from 35% to 50%, and phase B decreased from 65% to 50%.

[0065] The mobile phase flow rate was 1.0 mL / min, the column temperature was 30°C, the detector was an ultraviolet detector, and the detection wavelength was 267 nm.

[0066] Example 10:

[0067] The method for constructing the HPLC characteristic spectrum of the donkey-hide gelatin bone-strengthening oral liquid is carried out according to the following steps:

[0068] Preparation of the test solution: accurately measure 10 mL of donkey-hide gelatin bone strengthening oral liquid, add 50 mL of anhydrous ethanol and vortex until thoroughly mixed, sonicate for 1.2 h, filter, evaporate the filtrate in a water bath until nearly dry, add 20 mL of purified water to dissolve, extract with petroleum ether, discard the upper petroleum ether liquid, extract the lower water liquid with ethyl acetate, collect the ethyl acetate extract, evaporate to dryness in a water bath, dilute to 50 mL with methanol, shake well, and filter through 0.45 μm to obtain the test solution;

[0069] Preparation of mixed reference solution: Accurately weigh dangshenin, calycosin, calycosin, formononetin, and formononetin reference substance, and dilute to 50 mL with methanol to obtain a mixed reference solution containing 1.81 mg / mL dangshenin, 4.87 mg / mL calycosin, 7.66 mg / mL calycosin, 4.23 mg / mL formononetin, and 6.71 mg / mL formononetin.

[0070] Construction of characteristic spectrum: Accurately aspirate 10 μL of the mixed reference solution and the test solution respectively, inject them into the high performance liquid chromatograph to obtain the HPLC characteristic spectrum of the donkey-hide gelatin bone strengthening oral liquid.

[0071] Among them, the chromatographic conditions of the high performance liquid chromatography are:

[0072] The chromatographic column was an InertSustainAQ-C18 column, 250 mm × 4.6 mm, 5 μm;

[0073] The mobile phase is an acetonitrile-phosphoric acid system, in which phase A is acetonitrile and phase B is a 0.1% by mass phosphoric acid solution. Gradient elution is used, and the elution gradient includes:

[0074] From 0 min to 20 min, phase A increased from 10% to 25%, and phase B decreased from 90% to 75%;

[0075] From 20 to 40 minutes, phase A increased from 25% to 35%, and phase B decreased from 75% to 65%;

[0076] From 40 to 60 minutes, phase A increased from 35% to 50%, and phase B decreased from 65% to 50%.

[0077] The mobile phase flow rate was 1.0 mL / min, the column temperature was 30°C, the detector was an ultraviolet detector, and the detection wavelength was 267 nm.

[0078] Example 11:

[0079] Method for constructing HPLC characteristic spectrum of donkey-hide gelatin bone-strengthening oral liquid

[0080] Instruments and materials:

[0081] A Shimadzu LC-20AD high-performance liquid chromatograph was used, equipped with an online degasser, a quaternary pump, an automatic sampler, a column oven, and a UV detector. A CPA225D 1 / 100,000 electronic balance was purchased from Beijing Sartorius Scientific Instrument Co., Ltd. A GL-88B vortex mixer was purchased from Haimen Qilin Bell Instrument Manufacturing Co., Ltd. An 8302 constant-temperature water bath was purchased from Shanghai Lipu Automation Instrument Factory. A Smart-S15UV laboratory ultrapure water purifier was purchased from Shanghai Hetai Instrument Co., Ltd. An SK3300LHC ultrasonic cleaner was purchased from Shanghai Kedao Ultrasonic Instrument Co., Ltd. Methanol and acetonitrile were of chromatographic grade and were purchased from Fisher Scientific, USA. Petroleum ether, ethyl acetate, and anhydrous ethanol were of analytical grade. Ultrapure water was homemade.

[0082] Donkey-hide gelatin bone-strengthening oral liquid (batch numbers: 210415, 210522, 210603, 210720, 210822, 211020, 211103, 211222, 220102, 220407, 220510, 220512, 220523, 220524, 220531, numbers S1 to S15) were all provided by Xinjiang Huashidan Pharmaceutical Co., Ltd.; donkey-hide gelatin bone-strengthening oral liquid without astragalus and donkey-hide gelatin bone-strengthening oral liquid without codonopsis were both provided by Xinjiang Huashidan Pharmaceutical Co., Ltd.

[0083] The reference substances, calycosin (batch number 11920-201907) and codonopsis pilosula (batch number 11732-201908), were purchased from the China Food and Drug Inspection Institute; the reference substances, formononetin (batch number MUST-21080311), formononetin (batch number SF8070), and calycosin (batch number MUST-21101117), were purchased from Chengdu Desite Biotechnology Co., Ltd.; the mass fractions of all the above reference substances were ≥95%.

[0084] Preparation of the test solution: Accurately measure 10 mL of donkey-hide gelatin bone strengthening oral liquid, add 50 mL of anhydrous ethanol and vortex to mix thoroughly, sonicate for 1.2 h, filter, evaporate the filtrate in a water bath until almost dry, add 20 mL of purified water to dissolve, extract with petroleum ether, discard the upper petroleum ether liquid, extract the lower water liquid with ethyl acetate, collect the ethyl acetate extract, evaporate to dryness in a water bath, make up to the volume with methanol in a 50 mL volumetric flask, shake well, and filter through 0.45 μm to obtain the test solution.

[0085] Preparation of mixed reference solution: Accurately weigh dangshenin, calycosin, calycosin, formononetin, and formononetin reference substance, and dilute to volume with methanol in a 50 mL volumetric flask to obtain a mixed reference solution containing 1.81 mg / mL dangshenin, 4.87 mg / mL calycosin, 7.66 mg / mL calycosin, 4.23 mg / mL formononetin, and 6.71 mg / mL formononetin.

[0086] Preparation of negative sample solution: Take 10 mL of donkey-hide gelatin bone strengthening oral solution without astragalus and 10 mL of donkey-hide gelatin bone strengthening oral solution without codonopsis, and prepare the test solution according to the preparation method to obtain astragalus negative sample solution and codonopsis negative sample solution.

[0087] Construction of characteristic spectrum: Accurately aspirate 10 μl of mixed reference solution, astragalus negative sample solution, codonopsis negative sample solution and test solution respectively, inject into high performance liquid chromatography, record chromatogram within 60 minutes, import spectrum data into "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A version" for analysis, and obtain the characteristic spectrum of donkey-hide gelatin bone strengthening oral liquid.

[0088] Among them, the chromatographic conditions of the high performance liquid chromatography are:

[0089] The chromatographic column was an InertSustainAQ-C18 column, 250 mm × 4.6 mm, 5 μm;

[0090] The mobile phase is an acetonitrile-phosphoric acid system, in which phase A is acetonitrile and phase B is a 0.1% by mass phosphoric acid solution. Gradient elution is used, and the elution gradient includes:

[0091] From 0 min to 20 min, phase A increased from 10% to 25%, and phase B decreased from 90% to 75%;

[0092] From 20 to 40 minutes, phase A increased from 25% to 35%, and phase B decreased from 75% to 65%;

[0093] From 40 to 60 minutes, phase A increased from 35% to 50%, and phase B decreased from 65% to 50%.

[0094] The mobile phase flow rate was 1.0 mL / min, the column temperature was 30°C, the detector was an ultraviolet detector, and the detection wavelength was 267 nm.

[0095] The HPLC spectra of the mixed reference solution and the test solution obtained according to the method for constructing the HPLC characteristic spectrum of donkey-hide gelatin bone strengthening oral liquid in Example 11 of the present invention are as follows: Figure 1 As shown, the HPLC spectra of negative sample solutions (astragalus negative sample solution and codonopsis negative sample solution) are as shown Figure 2 As shown in the figure, the characteristic spectrum superposition of 15 batches of donkey-hide gelatin bone strengthening oral liquid is as follows Figure 3 As shown, through Figure 1 HPLC spectrum of the mixed reference solution in Figures 2-3Peak identification was performed, and the results showed that peak 1 was caerulein, peak 2 was codonopsis glycoside, peak 3 was formononetin, peak 4 was caerulein, and peak 5 was formononetin. Among them, peaks 1, 3, 4, and 5 were attributed to Astragalus membranaceus, and characteristic peak 2 was codonopsis glycoside, which was attributed to Codonopsis pilosula.

[0096] Example 12: Methodological Investigation

[0097] 1. Precision experiment

[0098] Donkey-hide gelatin bone strengthening oral solution (batch number: 220510) was prepared according to the method for preparing the test solution. The test solution was injected continuously 6 times and the peak area of ​​the chromatographic peak was recorded. Results: The relative retention time (min) of the chromatographic peaks of precision is shown in Table 1. As shown in Table 1, the relative retention time RSD of each common peak is less than 0.2%, and the peak area RSD is less than 2.46%, indicating that the instrument precision is good.

[0099] Repeatability experiments

[0100] Six test solutions of Donkey-hide Gelatin Bone Strengthening Oral Liquid (Batch No. 220510) were prepared in parallel according to the test solution preparation method. Each of the six test solutions was injected and the peak areas were recorded. Results: The reproducible chromatographic peak retention times (min) are shown in Table 2. As can be seen from Table 2, the relative retention time RSDs for the common peaks were <0.2%, and the peak area RSDs were <2.73%, indicating good reproducibility of the method.

[0101] Stability test

[0102] Donkey-hide gelatin bone strengthening oral solution (batch number: 220510) was prepared according to the test solution preparation method. The test solution was injected at 0h, 2h, 4h, 6h, 12h, and 24h, and the peak area was recorded. Results: The chromatographic peak retention time (min) of stability is shown in Table 3. As shown in Table 3, the relative retention time RSD of each common peak is less than 0.3%, and the peak area RSD is less than 2.44%, indicating that the test solution has good stability within 24h.

[0103] Table 1

[0104]

[0105] Table 2

[0106]

[0107] Table 3

[0108]

[0109] In summary, the HPLC characteristic spectrum obtained by the method for constructing the HPLC characteristic spectrum of the donkey-hide gelatin bone-strengthening oral liquid of the present invention has good peak shape, high separation, easy identification, high similarity, strong specificity, good stability, accuracy and reliability; the intrinsic quality of the donkey-hide gelatin bone-strengthening oral liquid can be controlled and evaluated more comprehensively, and can be used for the pharmaceutical quality standard and quality control of the donkey-hide gelatin bone-strengthening oral liquid.

[0110] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or removed according to actual needs to meet the requirements of different situations.

Claims

1. A method for constructing an HPLC characteristic spectrum of donkey-hide gelatin bone strengthening oral liquid, characterized in that: The following steps were followed: Preparation of the test solution: Accurately weigh donkey-hide gelatin bone strengthening oral liquid, add anhydrous ethanol to mix, sonicate, filter, concentrate the filtrate to near dryness, add purified water to dissolve, extract with petroleum ether, discard the upper petroleum ether liquid, extract the lower aqueous liquid with ethyl acetate, collect the ethyl acetate extract and concentrate to near dryness, dissolve with methanol, shake well, filter, and obtain the test solution; Preparation of the mixed reference solution: Accurately weigh dangshen glycoside, calycosin isoflavone glycoside, calycosin isoflavone, formononetin, and formononetin reference substance, mix, and dissolve with methanol to obtain a mixed reference solution; Construction of characteristic spectrum: Accurately aspirate the mixed reference solution and test solution respectively, inject them into high performance liquid chromatography, and obtain the HPLC characteristic spectrum of donkey-hide gelatin bone strengthening oral liquid; In the chromatographic conditions of the HPLC, the chromatographic column was a C18 column, 250 mm x 4.6 mm, 5 μm; In the chromatographic conditions of the high performance liquid chromatography, the mobile phase is an acetonitrile phosphoric acid system, in which phase A is acetonitrile and phase B is a phosphoric acid solution with a mass percentage concentration of 0.05% to 0.15%. Gradient elution is adopted, and the elution gradient includes: From 0 to 20 minutes, the concentration of phase A increased from 10% to 25%, and the concentration of phase B decreased from 90% to 75%. From 20 to 40 minutes, the concentration of phase A increased from 25% to 35%, and the concentration of phase B decreased from 75% to 65%. From 40 to 60 minutes, phase A increased from 35% to 50%, and phase B decreased from 65% to 50%; The detector is an ultraviolet detector with a detection wavelength of 267 nm.

2. The method for constructing the HPLC characteristic spectrum of donkey-hide gelatin bone strengthening oral liquid according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography were as follows: the mobile phase flow rate was 1.0 mL / min and the column temperature was 30 °C.

3. The method for constructing the HPLC characteristic spectrum of the donkey-hide gelatin bone strengthening oral liquid according to claim 1, characterized in that: In the preparation of the test solution, the volume ratio of donkey-hide gelatin bone strengthening oral solution to anhydrous ethanol is 1:4-6, the volume ratio of donkey-hide gelatin bone strengthening oral solution to purified water is 1:1-3, and the volume ratio of donkey-hide gelatin bone strengthening oral solution to methanol is 1:

5.

4. The method for constructing the HPLC characteristic spectrum of donkey-hide gelatin bone strengthening oral liquid according to claim 1, characterized in that: The concentrations of the reference substances in the mixed reference solution were 1.81 mg / mL of codonopsis pilosula, 4.87 mg / mL of calycosin, 7.66 mg / mL of calycosin, 4.23 mg / mL of formononetin, and 6.71 mg / mL of formononetin.

5. The method for constructing the HPLC characteristic spectrum of donkey-hide gelatin bone strengthening oral liquid according to claim 1, characterized in that: In the preparation of the test solution, the ultrasonic time is 1.0h to 1.5h.

Images