Method for constructing Weitai capsule intermediate fingerprint spectrum

Abstract

The application provides a construction method of Weitai capsule intermediate fingerprint, which comprises the following steps: preparation of reference substance solution, preparation of sample solution, precise suction of the sample solution and the reference substance solution, injection into a high performance liquid chromatograph, and recording of a chromatogram. The construction method of Weitai capsule intermediate fingerprint can detect multiple attribution peak components in the Weitai capsule intermediate, can detect multiple components in the Weitai capsule intermediate, and can be effectively used for evaluating the quality stability of the Weitai capsule intermediate product.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis technology, specifically relating to a method for constructing a fingerprint spectrum of an intermediate of Weitai capsules. Background Technology

[0002] Weitai capsules are made from eight medicinal herbs: gypsum, chebula, amomum, salvia miltiorrhiza, galangal, cyperus rotundus, sandalwood, and trogopterus xanthipes. They have the effects of warming the middle jiao and harmonizing the stomach, promoting qi circulation and relieving pain, and are suitable for stomach pain caused by spleen and stomach weakness and cold stagnation of qi. Current quality standards use thin-layer chromatography to identify tanshinone IIA and trogopterus xanthipes, but this cannot characterize the multiple components of the Weitai capsule intermediates, nor can it effectively evaluate the quality stability of the Weitai capsule intermediates. Therefore, it is essential to develop a method capable of detecting multiple components in the Weitai capsule intermediates to control their quality stability. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a method for constructing a fingerprint spectrum of a gastric capsule intermediate, which can simultaneously detect multiple components in the gastric capsule intermediate and can be effectively used to evaluate the product quality stability of the gastric capsule intermediate.

[0004] To achieve the above objectives, the present invention provides a method for constructing a fingerprint spectrum of an intermediate of a gastric capsule, comprising the following steps:

[0005] (1) Preparation of reference solution: Take appropriate amounts of rosmarinic acid, salvianolic acid B, tanshinone IIA, galangin, cryptotanshinone, α-cyperone, and cyperone, and dilute quantitatively with methanol;

[0006] (2) Preparation of test solution: Take an appropriate amount of the alcohol intermediate of Weitai capsules and place it in an Erlenmeyer flask. Add an appropriate amount of methanol solution, weigh it, and dissolve it by sonication. Cool it to room temperature, replenish the lost weight with methanol solution, shake well, filter through a 0.45 μm microporous membrane, and take the filtrate.

[0007] (3) Accurately pipette the test solution and reference solution into the high performance liquid chromatograph and record the chromatogram.

[0008] The chromatographic conditions of the high-performance liquid chromatograph are as follows:

[0009] Chromatographic column: C18 4.6mm × 250mm, 5μm;

[0010] Column temperature: 25~30℃;

[0011] Mobile phase A: Acetonitrile;

[0012] Mobile phase B: 0.05% phosphoric acid;

[0013] Detection wavelength: 279nm;

[0014] Flow rate: 0.8~1.2 ml / min;

[0015] The gradient elution procedure is as follows:

[0016] At 0 minutes, mobile phase A was 8% and mobile phase B was 92%.

[0017] At 10 minutes, mobile phase A was 15% and mobile phase B was 85%.

[0018] At 20 minutes, mobile phase A was 25% and mobile phase B was 75%.

[0019] At 30 minutes, mobile phase A was 25% and mobile phase B was 75%.

[0020] At 35 minutes, mobile phase A was 30% and mobile phase B was 70%.

[0021] At 37 minutes, mobile phase A was 40% and mobile phase B was 60%.

[0022] At 40 minutes, mobile phase A was 40% and mobile phase B was 60%.

[0023] At 45 minutes, mobile phase A was 55% and mobile phase B was 45%.

[0024] At 60 minutes, mobile phase A was 65% and mobile phase B was 35%.

[0025] At 65 minutes, mobile phase A was 70% and mobile phase B was 30%.

[0026] At 70 minutes, mobile phase A was 100% and mobile phase B was 0%.

[0027] At 75 minutes, mobile phase A was 100% and mobile phase B was 0%.

[0028] At 80 minutes, mobile phase A was 8% and mobile phase B was 92%.

[0029] At 90 minutes, mobile phase A was 8% and mobile phase B was 92%.

[0030] The methanol solution is an 80% vol aqueous methanol solution.

[0031] The ultrasonic power is 450~500W, the frequency is 35~45kHz, and the ultrasonic time is 25~35min.

[0032] The injection volume is 5~10 μl.

[0033] The intermediate of the Weitai capsule is the Weitai capsule alcohol extract powder. The preparation method of the Weitai capsule intermediate is as follows: take vinegar-processed Cyperus rotundus, Alpinia officinarum, Salvia miltiorrhiza, and Trogopterus xanthipes and reflux extract three times with 70% ethanol, combine the extracts, filter, concentrate the filtrate, dry and pulverize to obtain the product.

[0034] Compared with the prior art, the beneficial effect of the present invention is that it provides a method for constructing a fingerprint spectrum of the intermediate of the gastric capsule, which can detect the components of multiple assigned peaks in the intermediate of the gastric capsule, and can detect multiple components in the intermediate of the gastric capsule, and can be effectively used to evaluate the quality stability of the intermediate product of the gastric capsule. Attached Figure Description

[0035] Figure 1 This is the chromatogram of Example 2.

[0036] Figure 2 This is the chromatogram of Example 3.

[0037] Figure 3 This is the chromatogram of Example 4.

[0038] Figure 4 This is the chromatogram of Example 5. Detailed Implementation

[0039] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0040] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0041] Example 1: Preparation of intermediates for Weitai capsules

[0042] Take vinegar-processed Cyperus rotundus, Alpinia galanga, Salvia miltiorrhiza, and Trogopterus xanthipes, and extract them three times by reflux with ethanol: 4 hours for the first time, 3 hours for the second time, and 2 hours for the third time. Combine the extracts, filter them, concentrate the filtrate, dry it at 70°C, pulverize it, and pass it through an 80-mesh sieve to obtain the intermediate of Weitai capsules.

[0043] Example 2: Preliminary determination of chromatographic conditions

[0044] Preparation of reference solutions: Take appropriate amounts of rosmarinic acid, salvianolic acid B, tanshinone IIA, galangin, cryptotanshinone, α-cyperone, and cyperone, and add methanol to prepare solutions containing 200 µg per ml.

[0045] Preparation of the test solution: Take an appropriate amount of the intermediate of Weitai capsules, grind it into a fine powder, take about 1.0g, weigh it accurately, place it in a stoppered conical flask, accurately add 40ml of 80% methanol, weigh it, sonicate (power 500W, frequency 40kHz) for 30min, take it out and let it cool, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0046] Assay: Accurately pipette 10 μl of the reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0047] The chromatographic conditions were as follows: Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm); flow rate 1 ml / min; detection wavelength 279 nm. Mobile phase, column temperature, and injection volume are shown in Table 1; gradient elution programs are shown in Tables 2-5. The chromatogram is shown below. Figure 1 .

[0048] Table 1 Chromatographic conditions for each method

[0049]

[0050] Table 2 Gradient elution procedure for Method 1

[0051]

[0052] Table 3 Gradient elution procedure for Method 2

[0053]

[0054] Table 4 Gradient elution procedure for Method 3

[0055]

[0056] Table 5 Gradient elution procedure for Method 4

[0057]

[0058] Table 6 Gradient elution procedure for Method 5

[0059]

[0060] from Figure 1As can be seen, within the first 60 minutes of retention time, fingerprinting method 3 (mobile phase: methanol-0.05% phosphoric acid system) showed almost no peak response. The other methods, using acetonitrile-0.05% phosphoric acid system, displayed more chromatographic peak information and better peak parameters; therefore, acetonitrile-0.05% phosphoric acid was chosen as the mobile phase. Comparing fingerprinting method 2 (column temperature 25℃) and fingerprinting method 4 (column temperature 30℃), it is evident that at 30℃, the chromatographic baseline is flatter, making 30℃ the preferred column temperature. Comparing the chromatograms of fingerprinting methods 1, 2, 3, and 4 (injection volume 10 μl) and fingerprinting method 5 (injection volume 5 μl), it is clear that under fingerprinting method 5 conditions, the chromatographic baseline is relatively flat, the chromatographic peak distribution is uniform, and the chromatographic parameters are better; therefore, 5 μl is the preferred injection volume. In conclusion, fingerprinting method 5 was selected as the initial chromatographic condition for peak localization.

[0061] Example 3 Peak Positioning Test

[0062] Preparation of reference solutions: Take appropriate amounts of rosmarinic acid, salvianolic acid B, tanshinone IIA, galangin, cryptotanshinone, α-cyperone, and cyperone, and add methanol to prepare solutions containing 200 µg per ml.

[0063] Preparation of the test solution: Take about 1.0 g of the intermediate of Weitai capsules, place it in an Erlenmeyer flask, add 40 mL of 80% methanol aqueous solution, and weigh it. Sonicate (power 500 W, frequency 40 kHz) for 30 min, cool to room temperature, and weigh it again. Make up the weight loss with 80% methanol aqueous solution, shake well, filter through a 0.45 μm microporous membrane, and collect the filtrate.

[0064] Assay: Accurately pipette 5 µl each of the reference solution and the test solution into the liquid chromatograph and record the chromatogram.

[0065] Chromatographic conditions were as follows: Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm); acetonitrile as mobile phase A and 0.05% phosphoric acid as mobile phase B, using the gradient elution program in Table 6; flow rate 1 mL / min; column temperature 30 °C; detection wavelength 279 nm; injection volume 5 μL. The peak localization diagram is shown below. Figure 2 .

[0066] from Figure 2 As can be seen from the experimental results obtained under the initially determined chromatographic conditions, there is no corresponding chromatographic peak response within the retention time range of 28 min to 44 min, and the peak shape of some target peaks is not good.

[0067] Example 4: Gradient elution program optimization

[0068] Preparation of the test solution: Take about 1.0 g of the intermediate of Weitai capsules, place it in an Erlenmeyer flask, add 40 mL of 80% methanol aqueous solution, and weigh it. Sonicate (power 500 W, frequency 40 kHz) for 30 min, cool to room temperature, and weigh it again. Make up the weight loss with 80% methanol aqueous solution, shake well, filter through a 0.45 μm microporous membrane, and collect the filtrate.

[0069] Determination method: Accurately pipette 5µl of the test solution and inject it into the liquid chromatograph, and record the chromatogram.

[0070] The chromatographic conditions were as follows: Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm); acetonitrile as mobile phase A, 0.05% phosphoric acid as mobile phase B, with specific elution gradient programs shown in Tables 7-12; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 279 nm; injection volume 5 μL. The chromatogram is shown below. Figure 3 .

[0071] Table 7 Gradient elution program ①

[0072]

[0073] Table 8 Gradient elution program ②

[0074]

[0075] Table 9 Gradient elution program ③

[0076]

[0077] Table 10 Gradient elution program ④

[0078]

[0079] Table 11 Gradient elution program ⑤

[0080]

[0081] Table 12 Gradient elution program ⑥

[0082]

[0083] from Figure 3It can be seen that the separation effects of different gradient elution programs vary significantly. Gradient elution program ① failed to detect the target peak within the retention time range of 20–25 min. Gradient elution programs ②, ③, ④, and ⑥ showed little change in chromatograms within the retention time range of 20–50 min, failing to achieve the expected optimization, and compared to gradient elution program ⑤, the chromatographic peak distribution was less uniform, and the baseline was less stable. Although gradient elution program ⑥ had a shorter elution time, after a retention time of 30 min, more interfering peaks appeared compared to gradient elution program ⑤. In summary, gradient elution program ⑤ achieved the best performance in terms of uniform peak distribution, stable baseline, moderate peak capacity, and optimal chromatographic peak parameters.

[0084] Example 5: Characteristic Peak Determination

[0085] Preparation of reference solutions: Take appropriate amounts of rosmarinic acid, salvianolic acid B, tanshinone IIA, galangin, cryptotanshinone, α-cyperone, and cyperone, and add methanol to prepare solutions containing 200 µg per ml.

[0086] Preparation of the test solution: Take about 1.0 g of the intermediate of Weitai capsules, place it in an Erlenmeyer flask, add 40 mL of 80% methanol aqueous solution, and weigh it. Sonicate (power 500 W, frequency 40 kHz) for 30 min, cool to room temperature, and weigh it again. Make up the weight loss with 80% methanol aqueous solution, shake well, filter through a 0.45 μm microporous membrane, and collect the filtrate.

[0087] Assay: Accurately pipette 5 µL of the test solution and inject it into the liquid chromatograph, then record the chromatogram. See the chromatogram below. Figure 4 .

[0088] The chromatographic conditions were as follows: Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm); acetonitrile as mobile phase A and 0.05% phosphoric acid as mobile phase B, with the specific elution gradient program shown in Table 11; flow rate of 1.0 ml / min; column temperature of 30 ℃; detection wavelength of 279 nm; and injection volume of 5 μl.

[0089] like Figure 4 As shown, a total of 17 common peaks were identified, including peak 3, which is rosmarinic acid peak; peak 4, which is salvianolic acid B peak; peak 8, which is galangin peak; peak 13, which is cyperone peak; peak 14, which is cryptotanshinone peak; peak 15, which is α-cyperone peak; and peak 16, which is tanshinone IIA peak.

[0090] The above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a fingerprint spectrum of an intermediate of a gastric capsule, comprising the following steps: (1) Preparation of reference solution: Take appropriate amounts of rosmarinic acid, salvianolic acid B, tanshinone IIA, galangin, cryptotanshinone, α-cyperone, and cyperone, and dilute quantitatively with methanol; (2) Preparation of test solution: Take an appropriate amount of the intermediate of Weitai capsules and place it in an Erlenmeyer flask. Add an appropriate amount of methanol solution, weigh it, and dissolve it by sonication. Cool it to room temperature, replenish the lost weight with methanol solution, shake well, filter, and take the filtrate. (3) Accurately pipette the test solution and reference solution into the high performance liquid chromatograph and record the chromatogram; The chromatographic conditions of the high-performance liquid chromatograph are as follows: Chromatographic column: C18 4.6mm × 250mm, 5μm; Column temperature: 25~30℃; Mobile phase A: Acetonitrile; Mobile phase B: 0.05% phosphoric acid; Detection wavelength: 279nm; The mobile phase flow rate was 0.8~1.2 ml / min; The injection volume is 5~10 μl; The gradient elution procedure is as follows: At 0 minutes, mobile phase A was 8% and mobile phase B was 92%. At 10 minutes, mobile phase A was 15% and mobile phase B was 85%. At 20 minutes, mobile phase A was 25% and mobile phase B was 75%. At 30 minutes, mobile phase A was 25% and mobile phase B was 75%. At 35 minutes, mobile phase A was 30% and mobile phase B was 70%. At 37 minutes, mobile phase A was 40% and mobile phase B was 60%. At 40 minutes, mobile phase A was 40% and mobile phase B was 60%. At 45 minutes, mobile phase A was 55% and mobile phase B was 45%. At 60 minutes, mobile phase A was 65% and mobile phase B was 35%. At 65 minutes, mobile phase A was 70% and mobile phase B was 30%. At 70 minutes, mobile phase A was 100% and mobile phase B was 0%. At 75 minutes, mobile phase A was 100% and mobile phase B was 0%. At 80 minutes, mobile phase A was 8% and mobile phase B was 92%. At 90 minutes, mobile phase A was 8% and mobile phase B was 92%.

2. The method for constructing the fingerprint spectrum of the intermediate of the gastric capsule as described in claim 1, characterized in that, The methanol solution is an 80% vol aqueous methanol solution.

3. The method for constructing the fingerprint spectrum of the intermediate of the gastric capsule as described in claim 1, characterized in that, The ultrasonic power is 450~500W, the frequency is 35~45kHz, and the ultrasonic time is 25~35min.

4. The method for constructing the fingerprint spectrum of the intermediate of the gastric capsule as described in claim 1, characterized in that, The column temperature of the chromatographic column is 30℃.

5. The method for constructing the fingerprint spectrum of the intermediate of the gastric capsule as described in claim 1, characterized in that, The fingerprint spectrum of the intermediate of the Weitai capsule includes 17 characteristic peaks.

6. The method for constructing the fingerprint spectrum of the intermediate of the gastric capsule as described in claim 5, characterized in that, Among the 17 characteristic peaks, peak 3 is rosmarinic acid, peak 4 is salvianolic acid B, peak 8 is galangin, peak 13 is cyperone, peak 14 is cryptotanshinone, peak 15 is α-cyperone, and peak 16 is tanshinone IIA.

7. The method for constructing the fingerprint spectrum of the intermediate of the gastric capsule as described in any one of claims 1 to 6, characterized in that, The intermediate of the Weitai capsule is the Weitai capsule alcohol extract powder.

8. The method for constructing the fingerprint spectrum of the intermediate of the gastric capsule as described in claim 7, characterized in that, The preparation method of the Weitai capsule alcohol extract powder is as follows: take vinegar-processed Cyperus rotundus, Alpinia officinarum, Salvia miltiorrhiza, and Trogopterus xanthipes and reflux extract three times with 70% ethanol, combine the extracts, filter, concentrate the filtrate, dry and pulverize to obtain the product.

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