A method for determining the HPLC content of kaempferia galanga medicinal material decoction pieces, standard decoction and formula granules

Abstract

The application provides a construction method for HPLC content determination of Kaempferia galanga medicinal material decoction pieces, standard decoction and formula granules, comprising the following steps: A) dissolving and extracting raw materials of the test sample by using a solvent to obtain a test solution; B) determining the test solution by using high performance liquid chromatography to obtain HPLC characteristic chromatograms of the Kaempferia galanga medicinal material decoction pieces, the standard decoction and the formula granules; and the high performance liquid chromatography chromatographic conditions are as follows: a C 18 column is used as the chromatographic column; a mobile phase A is acetonitrile solution; a mobile phase B is 0.1% phosphoric acid aqueous solution; and gradient elution is adopted. The application adopts the high performance liquid chromatography, selects acetonitrile-0.1% phosphoric acid solution as the mobile phase for gradient elution, takes cinnamic acid, 4-methoxycinnamic acid and ethyl p-methoxycinnamate as reference substances, establishes an HPLC content determination method for the Kaempferia galanga medicinal material decoction pieces, the standard decoction and the formula granules, and provides more scientific technical means for controlling the medicinal quality of the Kaempferia galanga medicinal material decoction pieces, the standard decoction and the formula granules.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a method for constructing an HPLC method for determining the content of galangal medicinal slices, standard decoctions, and formulated granules. Background Technology

[0002] Kaempferia galanga L. is a perennial, low-growing herbaceous plant belonging to the ginger family and the Kaempferia genus. It has tuberous rhizomes, solitary or in clusters, and is aromatic. Leaves typically grow close to the ground, are nearly round, glabrous or sparsely covered with long, soft hairs on the underside, and sessile. Flowers are terminal, partially hidden within the leaf sheath; bracts are lanceolate; flowers are white, fragrant, and easily wither; the calyx is approximately the same length as the bracts; the lip is white; stamens lack filaments; the connective appendage is square; the fruit is a capsule.

[0003] Kaempferia galanga standard decoction is a freeze-dried powder made from the medicinal material after processing according to a fixed preparation process; Kaempferia galanga formula granules are formula granules made from Kaempferia galanga medicinal material after processing and according to the main quality indicators of the standard decoction. Both of the above are water-soluble extracts.

[0004] To ensure the uniformity and stability of the quality of galangal slices, standard decoctions, and formulated granules, a new characteristic chromatographic method is needed for quality control. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for determining the content of galangal medicinal slices, standard decoctions, and formula granules by HPLC. The method of the present invention is accurate and reliable.

[0006] A method for constructing an HPLC method for the determination of the content of galangal medicinal slices, standard decoctions, and formulated granules, comprising:

[0007] A) Dissolve and extract the raw material of the test sample using a solvent to obtain the test solution;

[0008] B) The test solution was determined by high performance liquid chromatography to obtain the HPLC characteristic chromatograms of galangal medicinal slices, standard decoctions, and formula granules;

[0009] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile solution, mobile phase B is 0.1% phosphoric acid aqueous solution, and gradient elution is used.

[0010] The raw materials for the test samples described in this invention are one or more of the following: galangal medicinal materials, galangal slices, galangal standard decoction, or galangal formula granules.

[0011] A method for determining the content of galangal medicinal materials in processed slices, standard decoctions, and formulated granules by HPLC is provided. First, the raw materials are dissolved and extracted with solvent to obtain the test solution.

[0012] When the raw materials are galangal medicinal materials and galangal slices, step A) involves decocting the galangal medicinal materials or galangal slices in water, filtering, dissolving the residue in 50% methanol, filtering, and obtaining the test solution; the ratio of the mass g of the test sample raw material, the volume mL of water, and the volume mL of 50% methanol is 2:50:10; the decoction time is 30 min.

[0013] When the raw material is galangal standard decoction or galangal formula granules, step A) is to treat the galangal standard decoction or galangal formula granules with 50% methanol using ultrasound, cool, filter, and obtain the test solution;

[0014] The ultrasound power is 600W, the frequency is 40kHz, and the ultrasound time is 30min.

[0015] The ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.4:25.

[0016] All the raw materials mentioned above can be subjected to quality control and qualitative and quantitative detection by the method of the present invention.

[0017] The present invention also includes the preparation of a reference solution: cinnamic acid, 4-methoxycinnamic acid and ethyl p-methoxycinnamate are dissolved in methanol to obtain a reference solution;

[0018] The reference solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the galangal medicinal slices, standard decoction, and formula granules were qualitatively determined based on the chromatogram of the reference.

[0019] The preferred concentrations of the reference solution in this invention are: cinnamic acid 25 μg / mL, 4-methoxycinnamic acid 50 μg / mL, and ethyl p-methoxycinnamate 10 μg / mL.

[0020] The test solution was analyzed by high performance liquid chromatography to obtain the HPLC characteristic chromatograms of galangal medicinal slices, standard decoctions, and formula granules.

[0021] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method are as follows: the chromatographic column is a C18 column; the chromatographic column has a size of 250×4.6mm and a diameter of 5μm; the column temperature is 5℃; under the above column temperature, the chromatographic peaks are symmetrical, the separation is good, and the peaks are eluted completely.

[0022] In this invention, mobile phase A is an acetonitrile solution, and mobile phase B is a 0.1% phosphoric acid aqueous solution, with gradient elution.

[0023] Specifically, the gradient elution is as follows:

[0024] 0–10 min, Phase A: 2%–10%, Phase B: 98–90%;

[0025] 10–30 min, Phase A: 10%–25%, Phase B: 90%–75%;

[0026] 30–40 min, Phase A: 25%–30%, Phase B: 75%–70%;

[0027] 40–50 min, Phase A: 30%–45%, Phase B: 70%–55%;

[0028] 50–60 min, Phase A: 45%–100%, Phase B: 55%–0%.

[0029] The theoretical plate number, calculated based on the cinnamic acid peak, should be no less than 5000.

[0030] The present invention exhibits good baseline separation, good peak separation, and stable baseline under the above-mentioned elution gradient.

[0031] The flow rate of the mobile phase described in this invention is 1.0 mL / min.

[0032] The present invention found that the chromatographic peaks were well separated and the resolution was moderate at the above flow rates, which is the optimal solution.

[0033] The injection volume was 10 μL.

[0034] The detection wavelength is 270 nm. The inventors have discovered that at 270 nm, the chromatographic peak information content is greater, the chromatographic baseline is more stable, and the peak areas are larger.

[0035] This invention uses a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine to evaluate the similarity of HPLC characteristic chromatograms of galangal medicinal slices, standard decoctions, and formula granules. The resulting standard HPLC characteristic chromatograms of galangal medicinal slices, standard decoctions, and formula granules consist of eight characteristic peaks, of which peak 5 (S): cinnamic acid; peak 6: 4-methoxycinnamic acid; and peak 8: ethyl p-methoxycinnamate.

[0036] In the characteristic spectrum of the kaempferia galanga formulation granules, cinnamic acid is used as the reference peak S. The relative retention time of each characteristic peak and peak S is calculated. The relative retention time is within ±10% of the specified value. The specified values ​​are: 0.25 (peak 1), 0.40 (peak 2), 0.62 (peak 3), 0.80 (peak 4), and 1.13 (peak 7).

[0037] In the characteristic spectrum of the kaempferia standard decoction, cinnamic acid was used as the reference peak S. The relative retention times of each characteristic peak and peak S were calculated. The relative retention times were within ±10% of the specified values, which were 0.26 (peak 1), 0.43 (peak 2), 0.65 (peak 3), 0.80 (peak 4), 1.14 (peak 7), and 1.28 (peak 8).

[0038] In the characteristic spectrum of the kaempferia galanga medicinal material or decoction pieces, cinnamic acid is used as the reference peak S. The relative retention time of each characteristic peak and peak S is calculated. The relative retention time is within ±10% of the specified value. The specified values ​​are: 0.27 (peak 1), 0.43 (peak 2), 0.65 (peak 3), 0.80 (peak 4), 1.14 (peak 7), and 1.28 (peak 8).

[0039] This invention provides a method for identifying characteristic chromatograms of galangal medicinal slices, standard decoctions, and formula granules. The method described above is used for detection, and the detection results are analyzed.

[0040] This invention provides a method for determining the HPLC content of galangal medicinal slices, standard decoctions, and formulated granules, comprising: A) dissolving and extracting the test sample raw material in a solvent to obtain a test solution; B) determining the test solution using high-performance liquid chromatography (HPLC) to obtain the HPLC characteristic chromatograms of galangal medicinal slices, standard decoctions, and formulated granules; the HPLC chromatographic conditions are as follows: a C18 column; mobile phase A is acetonitrile solution, mobile phase B is 0.1% phosphoric acid aqueous solution, and gradient elution is used. This invention employs HPLC, using acetonitrile-0.1% phosphoric acid solution as the mobile phase for gradient elution, and using cinnamic acid, 4-methoxycinnamic acid, and ethyl p-methoxycinnamate as references, to establish an HPLC method for determining the content of galangal medicinal slices, standard decoctions, and formulated granules, providing more scientific technical means for controlling the medicinal quality of galangal medicinal slices, standard decoctions, and formulated granules. Attached Figure Description

[0041] Figure 1 The results of the extraction method examination are shown in the figure;

[0042] Figure 2 Investigation of extraction solvents;

[0043] Figure 3 To investigate the amount of solvent added;

[0044] Figure 4 For the purpose of examining the extraction time;

[0045] Figure 5 Chromatographic peak identification;

[0046] Figure 6 Cinnamic acid spectrum - reference standard;

[0047] Figure 7 Cinnamic acid spectrum - medicinal materials;

[0048] Figure 8 Spectrum of 4-methoxycinnamic acid - reference standard;

[0049] Figure 9Spectrum of 4-methoxycinnamic acid - medicinal materials;

[0050] Figure 10 Spectrum of ethyl p-methoxycinnamate - reference standard;

[0051] Figure 11 Spectrum of ethyl p-methoxycinnamate - medicinal material;

[0052] Figure 12 Different instruments were used for the investigation.

[0053] Figure 13 Investigation of different chromatographic columns.

[0054] Figure 14 Atlas of the characteristics of Kaempferia galanga medicinal materials;

[0055] Figure 15 Atlas of the characteristics of Kaempferia galanga medicinal materials;

[0056] Figure 16 A comparative characteristic atlas of Kaempferia galanga medicinal materials;

[0057] Figure 17 Characteristic Atlas of Kaempferia galanga Medicinal Slices;

[0058] Figure 18 Characteristic Atlas of Kaempferia galanga Medicinal Slices;

[0059] Figure 19 A comparative atlas of galangal slices;

[0060] Figure 20 Chromatographic peak identification;

[0061] Figure 21 Cinnamic acid spectrum - reference standard;

[0062] Figure 22 Cinnamic acid spectrum - standard decoction;

[0063] Figure 23 Spectrum of 4-methoxycinnamic acid - reference standard;

[0064] Figure 24 Spectrum of 4-methoxycinnamic acid - standard decoction;

[0065] Figure 25 Spectrum of ethyl p-methoxycinnamate - reference standard;

[0066] Figure 26 Spectrum of ethyl p-methoxycinnamate - standard decoction;

[0067] Figure 27 Exploration using different instruments;

[0068] Figure 28 Investigation of different chromatographic columns.

[0069] Figure 29 Characteristic spectrum of standard decoction of galangal;

[0070] Figure 30 Characteristic spectrum of standard decoction of galangal;

[0071] Figure 31 Compare the characteristic maps.

[0072] Figure 32 Chromatograms of kaempferia galanga granules at different wavelengths.

[0073] Figure 33 Mobile phase selection;

[0074] Figure 34 Column temperature investigation.

[0075] Figure 35 Flow velocity study.

[0076] Figure 36 Delayed investigation;

[0077] Figure 37 Extraction methods were examined.

[0078] Figure 38 Investigation of extraction solvents.

[0079] Figure 39 Investigation of the amount of solvent added.

[0080] Figure 40 Extraction time was examined.

[0081] Figure 41 For chromatographic peak identification;

[0082] Figure 42 Spectrum of 4-methoxycinnamic acid - reference standard;

[0083] Figure 43 Spectrum of 4-methoxycinnamic acid - Kaempferia galanga granules;

[0084] Figure 44 Cinnamic acid spectrum - reference standard;

[0085] Figure 45 Cinnamic acid spectrum - Kaempferia galanga granules;

[0086] Figure 46 Spectrum of ethyl p-methoxycinnamate - reference standard;

[0087] Figure 47 Spectrum of ethyl p-methoxycinnamate - Kaempferia galanga granules.

[0088] Figure 48 Different instruments were used for the investigation.

[0089] Figure 49 Investigation of different chromatographic columns.

[0090] Figure 50 Characteristic spectrum of galangal formulation granules;

[0091] Figure 51 Compare the characteristic maps. Detailed Implementation

[0092] This invention provides a method for the HPLC content determination of galangal medicinal slices, standard decoctions, and formulated granules. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0093] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for constructing HPLC content determination of galangal medicinal slices, standard decoctions, and formula granules provided by the present invention.

[0094] Example 1: UPLC Characteristic Map of Kaempferia galanga

[0095] 1. Instruments and Materials

[0096] High-performance liquid chromatographs: Agilent 1260 high-performance liquid chromatograph; Waters H-class plus ultra-high-performance liquid chromatograph; Thermo Fisher Vanquish Core high-performance liquid chromatograph;

[0097] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0098] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0099] Ultrasonic cleaner: KQ600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0100] Chromatographic columns: Column 1 (Polar-Phenyl 120A, 4.6*250mm, 5μm); Column 2 (SVEATMC18Opal, 4.6*250mm, 5μm); Column 3 (Chromsil™ C18, 4.6*250mm, 5μm). Acetonitrile and phosphoric acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0101] 4-Methoxycinnamic acid (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq22061001, purity 99.35%);

[0102] Cinnamic acid (China National Institutes for Food and Drug Control, batch number: 110786-202305, purity: 99.8%);

[0103] Ethyl p-methoxycinnamate (China National Institutes for Food and Drug Control, batch number: 110835-202005, purity: 99.4%);

[0104] Kaempferia galanga reference material (China National Institutes for Food and Drug Control, batch number: 121504-201203)

[0105] Kaempferia galanga (purchased by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: SN-01, SN-02, SN-03, SN-04, SN-05, SN-06, SN-07, SN-08, SN-09, SN-10, SN-11, SN-12, SN-13, SN-14, SN-15, SN-16, SN-17)

[0106] Kaempferia galanga slices (prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: SN-YP01, SN-YP02, SN-YP03, SN-YP04, SN-YP05, SN-YP06, SN-YP07, SN-YP08, SN-YP09, SN-YP10, SN-YP11, SN-YP12, SN-YP13, SN-YP14, SN-YP15, SN-YP16, SN-YP17)

[0107] Characteristic spectrum determination method

[0108] Based on the methodology of the characteristic chromatogram analysis of standard decoctions of Kaempferia galanga, the following method for analyzing the characteristic chromatograms of Kaempferia galanga is proposed:

[0109] 2. Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5.0 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 1.0 mL / min; the column temperature was 35℃; and the detection wavelength was 270 nm. The theoretical plate number, calculated based on the cinnamic acid peak, should not be less than 5000.

[0110]

[0111]

[0112] 2.1 Preparation of Reference Solutions: Take 2g of *Kaempferia galanga* reference material, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 10ml of 50% methanol, shake well, filter, and use the filtrate as the reference solution. Separately, accurately weigh appropriate amounts of cinnamic acid and 4-methoxycinnamic acid reference standards, and prepare mixed solutions containing 25μg of cinnamic acid and 50μg of 4-methoxycinnamic acid per 1ml using methanol, as mixed reference solutions. Finally, accurately weigh an appropriate amount of ethyl p-methoxycinnamate reference standard, and prepare a solution containing 10μg of ethyl p-methoxycinnamate per 1ml using methanol, as the reference solution.

[0113] 2.2 Preparation of the test solution: Take 2g of Kaempferia galanga powder (crushed and passed through a No. 2 sieve before use), place it in a stoppered conical flask, and prepare it according to the preparation method of the reference medicinal material. Take the filtrate to obtain the test solution.

[0114] 2.3 Determination Method: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0115] 2.3.1 Chromatographic conditions and system suitability test

[0116] The characteristic chromatographic conditions and system adaptability, as well as the characteristic chromatographic methods for particles, are consistent with those for standard decoctions.

[0117] 2.3.2 Preparation of the test solution

[0118] 2.3.2.1 Examination of Extraction Methods

[0119] Take 2g of Kaempferia galanga powder (batch number: SN-01), place it in a stoppered conical flask, add 10ml of 50% methanol, seal tightly, and extract ultrasonically for 30 minutes. Cool, shake well, filter, and collect the filtrate. Separately, place the powder in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 10ml of 50% methanol, shake well, filter, and collect the filtrate. See [link to other ingredients]. Figure 1 . Figure 1 The results of the extraction method evaluation are shown in the figure. The results indicate that, compared with ultrasonic extraction, the decoction extraction results are more consistent with the standard decoction control spectrum, and decoction extraction is the preferred method.

[0120] 2.3.2.2 Investigation of Extraction Solvents

[0121] Take 2.0g of Kaempferia galanga powder (batch number: SN-01), place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, shake well, filter, and dissolve the filtrate and residue in 10ml each of water, methanol, 70% methanol, and 50% methanol respectively, shake well, filter, and collect the filtrate. The results are shown in the figure. Figure 2 . Figure 2Extraction solvent investigation; the results showed that the extraction efficiency was higher when the extraction solvent was 50% methanol, and the extraction solvent was set at 50% methanol.

[0122] 2.3.2.3 Investigation of Solvent Addition Amount

[0123] Take 2.0g of Kaempferia galanga powder (batch number: SN-01), place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 10ml and 25ml of 50% methanol respectively, shake well, filter, and collect the filtrate to obtain the final product. Results are shown below. Figure 3 . Figure 3 Solvent addition amount investigation. The results showed that when the extraction solvent addition amount was 10 ml, the peak shape and resolution of each chromatographic peak were good, and the peak size was appropriate. Therefore, the solvent volume was selected as 10 ml.

[0124] 2.3.2.4 Examination of extraction time

[0125] Take 2.0g of Kaempferia galanga powder (batch number: SN-01), place it in a stoppered conical flask, add 50ml of water, decoct for 30-60 minutes, shake well, filter, dissolve the residue in 10ml of 50% methanol, shake well, filter, and collect the filtrate. Results are shown below. Figure 4 . Figure 4 Extraction time was investigated. The results showed that complete extraction was achieved at an extraction time of 30 minutes. Therefore, the extraction time was determined to be 30 minutes.

[0126] In summary, the preparation method of the Kaempferia galanga test solution is determined as follows: Take 2g of the powder (crushed and passed through a No. 2 sieve before use), place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, shake well, filter, evaporate the filtrate to dryness, add 10ml of 50% methanol to dissolve the residue, shake well, filter, and take the filtrate to obtain the solution.

[0127] 2.3.2.5 Methodological Examination

[0128] 2.3.2.5.1 Chromatographic Peak Identification

[0129] Preparation of the test solution: Prepare the Kaempferia galanga test solution according to the experimental conditions proposed above.

[0130] Preparation of reference solutions: Take 2g of Kaempferia galanga reference material, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, shake well, filter, evaporate to dryness, add 10ml of 50% methanol to dissolve, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of cinnamic acid, 4-methoxycinnamic acid, and ethyl p-methoxycinnamate reference standards, accurately weigh them, and add methanol to prepare mixed solutions containing 25μg of cinnamic acid, 50μg of 4-methoxycinnamic acid, and 10μg of ethyl p-methoxycinnamate per 1ml, respectively, as the reference solutions.

[0131] Preparation of negative control solution: Prepare negative control solution of Kaempferia galanga medicinal material according to the experimental conditions proposed above.

[0132] The characteristic peaks of Kaempferia galanga medicinal material were located. (See...) Figure 5-11 . Figure 5 Chromatographic peak identification; Figure 6 Cinnamic acid spectrum - reference standard; Figure 7 Cinnamic acid spectrum - medicinal materials; Figure 8 Spectrum of 4-methoxycinnamic acid - reference standard; Figure 9 Spectrum of 4-methoxycinnamic acid - medicinal materials; Figure 10 Spectrum of ethyl p-methoxycinnamate - reference standard; Figure 11 Spectrum of ethyl p-methoxycinnamate - medicinal material. Results showed that peak 5 (S) was cinnamic acid, peak 6 was 4-methoxycinnamic acid, and peak 7 was ethyl p-methoxycinnamate. In the following methodological investigation, eight characteristic peaks in the sample were examined.

[0133] 2.3.2.5.2 Precision Test

[0134] Take the test solution of Kaempferia galanga (batch number: SN-01), and inject it 6 times consecutively according to the proposed experimental method, 10 μl each time, and calculate the retention time of each characteristic peak. See Table 1.

[0135] Table 1 Precision test - retention time

[0136]

[0137] The results showed that the retention time RSD of each characteristic peak was 0.01% to 0.13%, indicating that the method had good precision.

[0138] 2.3.2.5.3 Repeatability Test

[0139] Six portions of Kaempferia galanga (batch number: SN-01) were accurately weighed and prepared and measured according to the proposed experimental method. The relative retention times of each characteristic peak were calculated. See Table 6-7.

[0140] Table 2 Repeatability Tests - Relative Retention Time

[0141]

[0142] The results showed that the relative retention time RSD of the six samples was 0.00%–0.45%, indicating that the method had good repeatability.

[0143] 2.3.2.5.4 Intermediate Precision Examination

[0144] 2.3.2.5.4.1 Investigation with different instruments

[0145] Based on the above-planned experimental conditions, test solutions were prepared from Kaempferia galanga (batch number: SN-01) and measured on Thermo Fisher Vanquish Core (instrument 1); Agilent 1260 (instrument 2); and Waters e2695 (instrument 3), respectively. See [link to relevant documentation]. Figure 12 Table 3. Figure 12 Different instruments were used for the investigation.

[0146] Table 3 Instrument Durability Test - Relative Retention Time

[0147]

[0148]

[0149] The results showed that the RSD of the relative retention time of each characteristic peak when the test sample was detected by the above three instruments was 0.72% to 6.93%, indicating that the instrument durability of this method was good.

[0150] 2.3.2.5.4.2 Investigations by different personnel and at different times

[0151] Based on the experimental conditions outlined above, different personnel (A and B) precisely weighed Kaempferia galanga (batch number: SN-01) at different times (T1 and T2) to prepare test samples, and then measured and calculated the relative retention times of each characteristic peak. See Table 4.

[0152] Table 4. Personnel and Time Assessment - Relative Retention Time

[0153]

[0154] The results show that the intermediate precision of the method is good when measured by different personnel at different times.

[0155] 2.3.2.5.5 Durability Assessment

[0156] 2.3.2.5.5.1 Column robustness test

[0157] Based on the above-specified experimental conditions, analyses were conducted using chromatographic columns 1 (Polar-Phenyl 120A, 4.6*250mm, 5μm), 2 (SVEATM C18 Opal, 4.6*250mm, 5μm), and 3 (ChromsilTM C18, 4.6*250mm, 5μm), respectively. The results are shown in the table below. Figure 13 Table 5. Figure 13 Investigation of different chromatographic columns.

[0158] Table 5. Column robustness study - relative retention time

[0159]

[0160] The results showed that when the samples were detected using the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks ranged from 0.72% to 6.93%, indicating that the columns used in this method had good robustness.

[0161] 2.3.2.5.5.2 Stability Assessment

[0162] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 6h, 12h, and 24h. See Table 6.

[0163] Table 6 Stability Study - Retention Time

[0164]

[0165] The results showed that the RSD of the retention time of the characteristic peak was between 0.01% and 0.50%, indicating that the sample solution was stable within 24 hours.

[0166] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The above eight characteristic peaks will be included in subsequent investigations.

[0167] 2.4 Determination of characteristic peaks and establishment of reference spectra

[0168] 2.4.1 Verification results of 17 batches of Kaempferia galanga medicinal materials

[0169] Using this method, characteristic spectral analysis was performed on 20 batches of samples to calculate the relative retention time.

[0170] .See Figure 23-24 Table 7. Figure 14 Characteristic spectrum of Kaempferia galanga medicinal material; Peak 5 (S): Cinnamic acid; Peak 6: 4-Methoxycinnamic acid; Peak 8: Ethyl p-methoxycinnamate (from bottom to top: SN-01, SN-02, SN-03, SN-04, SN-05, SN-06, SN-07, SN-08).

[0171] Figure 15 Characteristic spectrum of Kaempferia galanga medicinal material; Peak 5 (S): Cinnamic acid; Peak 6: 4-Methoxycinnamic acid; Peak 8: Ethyl p-methoxycinnamate; (from bottom to top: SN-09, SN-10, SN-11, SN-12, SN-13, SN-14, SN-15, SN-16, SN-17).

[0172] Table 7. Relative retention time of galangal medicinal materials

[0173]

[0174] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peak values, eight peaks with good robustness were selected as characteristic peaks. According to the methodological investigation results and the validation results of 17 batches of medicinal materials, the theoretical plate number, calculated based on the cinnamic acid peak, is tentatively set to be no less than 5000.

[0175] 2.4.2 Establishment of Limits for Relative Retention Time

[0176] Table 8 summarizes the methodological examination items and validation results:

[0177] Table 8 Summary of RSD% for Methodological Results—Relative Retention Time / Retention Time

[0178]

[0179]

[0180] The relative retention times of each characteristic peak were stable, and the relative retention times of other tested items were all within ±10% of the average value. The specified range for the relative retention times of each peak is tentatively set at ±10%.

[0181] To better reflect the transfer of values, and to maintain consistency with the relative retention times of the galangal granule formulation, the final specification stipulates that the test sample chromatogram should show 8 characteristic peaks, corresponding to the retention times of the 8 characteristic peaks in the reference chromatogram of the medicinal material. Peaks 5, 6, and 8 should correspond to the retention times of their respective reference peaks. The peak corresponding to the cinnamic acid reference peak is designated as peak S. The relative retention times of the remaining characteristic peaks and peak S should be calculated and should be within ±10% of the specified values. The specified values ​​are: 0.27 (peak 1), 0.43 (peak 2), 0.65 (peak 3), 0.80 (peak 4), 1.14 (peak 7), and 1.28 (peak 8).

[0182] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize chromatograms of 17 batches of Kaempferia galanga medicinal materials, and a reference chromatogram of the characteristic chromatograms of Kaempferia galanga medicinal materials was established. (See...) Figure 16 . Figure 16 Kaempferia galanga medicinal material reference characteristic spectrum; Peak 5 (S): cinnamic acid; Peak 6: 4-methoxycinnamic acid; Peak 8: ethyl p-methoxycinnamate

[0183] In summary, the feature map method is as follows:

[0184] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5.0 μm); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 270 nm. The theoretical plate number, calculated based on the cinnamic acid peak, should be no less than 5000.

[0185]

[0186] Preparation of the reference solution: Take 2g of Kaempferia galanga reference material, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 10ml of 50% methanol, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of cinnamic acid and 4-methoxycinnamic acid reference standards, accurately weigh them, and add methanol to prepare mixed solutions containing 25μg of cinnamic acid and 50μg of 4-methoxycinnamic acid per ml, respectively, as the reference solution; then take an appropriate amount of ethyl p-methoxycinnamate reference standard, accurately weigh it, and add methanol to prepare a solution containing 10μg of ethyl p-methoxycinnamate per ml, as the reference solution.

[0187] Preparation of the test solution: Take about 2g of the powder of this product (crushed and passed through a No. 2 sieve before use) and prepare the test solution together with the reference solution of the control medicinal material.

[0188] The assay method involves precisely pipetting 10 μl each of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0189] The chromatogram of the test sample should show eight characteristic peaks, and the retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. Peaks 5 and 6 should correspond to the retention times of their respective reference peaks. The peak corresponding to the cinnamic acid reference peak is designated as the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.27 (peak 1), 0.43 (peak 2), 0.65 (peak 3), 0.80 (peak 4), 1.14 (peak 7), and 1.28 (peak 8).

[0190] Example 2: Characteristic Atlas of Kaempferia galanga Slices

[0191] Synthetic comparison chart of 17 batches of galangal slices: Figure 17Characteristic chromatogram of galangal slices; Peak 5 (S): Cinnamic acid; Peak 6: 4-Methoxycinnamic acid; Peak 8: Ethyl p-methoxycinnamate (from bottom to top: SN-YP01, SN-YP02, SN-YP03, SN-YP04, SN-YP05, SN-YP06, SN-YP07, SN-YP08). Figure 18 Characteristic chromatogram of galangal slices; Peak 5 (S): Cinnamic acid; Peak 6: 4-Methoxycinnamic acid; Peak 8: Ethyl p-methoxycinnamate; (from bottom to top: SN-YP 09, SN-YP 10, SN-YP 11, SN-YP 12, SN-YP 13, SN-YP 14, SN-YP 15, SN-YP 16, SN-YP 17).

[0192] Table 9. Relative Retention Time of Kaempferia galanga Slices

[0193]

[0194] The relative retention times of each characteristic peak were stable, and the relative retention times of other tested items were all within ±10% of the average value. The specified range for the relative retention times of each peak is tentatively set at ±10%.

[0195] The final specification stipulates that the chromatogram of the test sample should show eight characteristic peaks, and the retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. Peaks 5 and 6 should correspond to the retention times of their respective reference peaks. The peak corresponding to the cinnamic acid reference peak is designated as the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.27 (peak 1), 0.43 (peak 2), 0.65 (peak 3), 0.80 (peak 4), 1.14 (peak 7), and 1.28 (peak 8).

[0196] The characteristic chromatograms of 17 batches of Kaempferia galanga slices were compared using the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version). See [link / reference]. Figure 19 . Figure 19 Comparative chromatogram of galangal slices; Peak 5 (S): cinnamic acid; Peak 6: 4-methoxycinnamic acid; Peak 8: ethyl p-methoxycinnamate.

[0197] In summary, the feature map method is as follows:

[0198] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5.0 μm); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 270 nm. The theoretical plate number, calculated based on the cinnamic acid peak, should be no less than 5000.

[0199]

[0200]

[0201] Preparation of the reference solution: Take 2g of Kaempferia galanga reference material, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 10ml of 50% methanol, shake well, filter, and use the filtrate as the reference solution. Separately, take appropriate amounts of cinnamic acid and 4-methoxycinnamic acid reference standards, accurately weigh them, and add methanol to prepare mixed solutions containing 25μg of cinnamic acid and 50μg of 4-methoxycinnamic acid per ml, respectively, as the reference solution. Then, take an appropriate amount of ethyl p-methoxycinnamate reference standard, accurately weigh it, and add methanol to prepare a solution containing 10μg of ethyl p-methoxycinnamate per ml, as the reference solution.

[0202] Preparation of the test solution: Take 2g of the powder of this product (crushed and passed through a No. 2 sieve before use), place it in a stoppered conical flask, and prepare it according to the preparation method of the reference medicinal material. Take the filtrate to obtain the test solution.

[0203] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0204] To better reflect the transfer of values, and to maintain consistency with the relative retention times of the galangal granule formulation, the final specification stipulates that the test sample chromatogram should show 8 characteristic peaks, corresponding to the retention times of the 8 characteristic peaks in the reference chromatogram of the medicinal material. Peaks 5 and 6 should correspond to the retention times of their respective reference peaks. The peak corresponding to the cinnamic acid reference peak is designated as peak S. The relative retention times of the remaining characteristic peaks and peak S should be calculated and should be within ±10% of the specified values. The specified values ​​are: 0.27 (peak 1), 0.43 (peak 2), 0.65 (peak 3), 0.80 (peak 4), 1.14 (peak 7), and 1.28 (peak 8).

[0205] Example 3: HPLC Characteristic Chromatography of Kaempferia galanga Standard Decoction

[0206] 3.1 Experimental Instruments and Materials

[0207] High-performance liquid chromatographs: Agilent 1260 HPLC system; Waters e-2695 HPLC system; Thermo Fisher Vanquish Core HPLC system;

[0208] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0209] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0210] Ultrasonic cleaner: KQ600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0211] Chromatographic column:

[0212] Column 1 (Polar-Phenyl 120A, 4.6*250mm, 5μm); Column 2 (SVEA™ C18 Opal, 4.6*250mm, 5μm); Column 3 (Chromsil™ C18, 4.6*250mm, 5μm)

[0213] Acetonitrile and phosphoric acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0214] 4-Methoxycinnamic acid (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq22061001, purity 99.35%);

[0215] Cinnamic acid (China National Institutes for Food and Drug Control, batch number: 110786-202305, purity: 99.8%);

[0216] Ethyl p-methoxycinnamate (China National Institutes for Food and Drug Control, batch number: 110835-202005, purity: 99.4%);

[0217] Kaempferia galanga reference material (China National Institutes for Food and Drug Control, batch number: 121504-201203)

[0218] Kaempferia galanga standard decoction (prepared by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: SN-BT01, SN-BT02, SN-BT03, SN-BT04, SN-BT05, SN-BT06, SN-BT07, SN-BT08, SN-BT09, SN-BT10, SN-BT11, SN-BT12, SN-BT13, SN-BT14, SN-BT15, SN-BT16, SN-BT17)

[0219] 3.2 Drafting Method

[0220] 3.2.1 Chromatographic conditions

[0221] The column was packed with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5.0 μm particle size); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 1.0 mL / min; the column temperature was 35 °C; and the detection wavelength was 270 nm. The theoretical plate number, calculated based on the cinnamic acid peak, should be no less than 5000.

[0222]

[0223] 3.2.2 Preparation of the reference solution

[0224] Take 2g of galangal reference material, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 10ml of 50% methanol, shake well, filter, and take the filtrate as the reference solution. Separately, accurately weigh appropriate amounts of cinnamic acid and 4-methoxycinnamic acid reference standards, add methanol to prepare a mixed solution containing 25μg of cinnamic acid and 50μg of 4-methoxycinnamic acid per ml, as the reference solution; then accurately weigh an appropriate amount of ethyl p-methoxycinnamate reference standard, add methanol to prepare a solution containing 10μg of ethyl p-methoxycinnamate per ml, as the reference solution.

[0225] 3.2.3 Preparation of the test solution

[0226] Take 0.4g of this product, place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product.

[0227] 3.2.4 Determination Method

[0228] Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0229] 3.2.4.1 Chromatographic conditions and system suitability test

[0230] The standard decoction and the finished granules have the same basic material composition, and the characteristic chromatographic conditions, system adaptability, and granule characteristic chromatographic methods are consistent.

[0231] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5.0 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 1.0 mL / min; the column temperature was 35℃; and the detection wavelength was 270 nm. The theoretical plate number, calculated based on the cinnamic acid peak, should not be less than 5000.

[0232]

[0233] 3.2.4.2 Investigation on the preparation of the test solution

[0234] The standard decoction and the finished granules have the same basic material composition, and the preparation method of the test solution is consistent with that of the finished granules.

[0235] The test solution is prepared as follows: Take 0.4g of this product, place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0236] 3.2.5 Methodological Examination

[0237] 3.2.5.1 Chromatographic Peak Identification

[0238] Preparation of the test solution: Prepare the standard decoction test solution of kaempferia galanga according to the experimental conditions proposed above.

[0239] Preparation of reference solutions: Take 2g of Kaempferia galanga reference material, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, shake well, filter, evaporate to dryness, add 10ml of 50% methanol to dissolve, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of cinnamic acid, 4-methoxycinnamic acid, and ethyl p-methoxycinnamate reference standards, accurately weigh them, and add methanol to prepare mixed solutions containing 25μg of cinnamic acid, 50μg of 4-methoxycinnamic acid, and 10μg of ethyl p-methoxycinnamate per 1ml, respectively, as the reference solutions.

[0240] Preparation of negative control solution: Prepare negative control solution of kaempferia galanga standard decoction according to the experimental conditions proposed above.

[0241] The characteristic peaks of the standard decoction of Kaempferia galanga were located. (See...) Figure 20-26 . Figure 20 Chromatographic peak identification; Figure 21 Cinnamic acid spectrum - reference standard; Figure 22 Cinnamic acid spectrum - standard decoction; Figure 23 4-Methoxycinnamic acid spectrum - reference standard; Figure 244 4-Methoxycinnamic acid spectrum - standard decoction; Figure 25Spectrum of ethyl p-methoxycinnamate - reference standard; Figure 26 Spectrum of ethyl p-methoxycinnamate - standard decoction. Results showed that peak 5 (S) was cinnamic acid, peak 6 was 4-methoxycinnamic acid, and peak 8 was ethyl p-methoxycinnamate. The following methodological investigation investigated these eight characteristic peaks in the sample.

[0242] 3.2.5.2 Precision Test

[0243] Take the test solution of Kaempferia galanga standard decoction (batch number: SN-BT01), and inject it 6 times consecutively according to the proposed experimental method, 10 μl each time, and calculate the retention time of each characteristic peak.

[0244] See Table 10.

[0245] Table 10 Precision Examination - Retention Time

[0246]

[0247] The results showed that the retention time RSD of each characteristic peak was 0.02% to 0.11%, indicating that the instrument has good precision.

[0248] 3.2.5.3 Repeatability Test

[0249] Accurately weigh 6 portions of the standard decoction of Kaempferia galanga (batch number: SN-BT01), prepare and measure it according to the proposed experimental method, and calculate the relative retention time of each characteristic peak. See Table 11.

[0250] Table 11 Repeatability Tests - Relative Retention Time

[0251]

[0252] The results showed that the relative retention time RSD of the six samples was 0%–0.36%, indicating that the method had good reproducibility.

[0253] 3.2.5.4 Intermediate Precision Examination

[0254] 3.2.5.4.1 Investigation with different instruments

[0255] Based on the above-planned experimental conditions, standard decoction of Kaempferia galanga (batch number: SN-BT01) was weighed to prepare test solutions, which were then measured on Thermo Fisher Vanquish Core (instrument 1); Agilent 1260 (instrument 2); and Waters e2695 (instrument 3), respectively. See [link to relevant documentation]. Figure 27 Table 12. Figure 27 Different instruments were used for the investigation.

[0256] Table 12 Instrument Durability Test - Relative Retention Time

[0257]

[0258] The results showed that when the test samples were detected using the above three instruments, the RSD of the relative retention time of each characteristic peak was 0.72% to 7.70%, indicating that the instruments used in this method have good durability.

[0259] 3.2.5.4.2 Investigations by different personnel and at different times

[0260] Based on the experimental conditions outlined above, different personnel (A and B) precisely weighed Kaempferia galanga standard decoction (batch number: SN-BT01) at different times (T1 and T2) to prepare test samples, and then measured and calculated the relative retention times of each characteristic peak. See Table 13.

[0261] Table 13 Personnel and Time Assessment - Relative Retention Time

[0262]

[0263] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0%-2.19%, indicating that the method had good intermediate precision.

[0264] 3.2.5.4.3 Durability Assessment

[0265] 3.2.5.4.3.1 Column robustness test

[0266] Based on the above-specified experimental conditions, analyses were conducted using chromatographic columns 1 (Polar-Phenyl 120A, 4.6*250mm, 5μm), 2 (SVEATM C18 Opal, 4.6*250mm, 5μm), and 3 (ChromsilTM C18, 4.6*250mm, 5μm), respectively. The results are shown in [Figure number missing]. Figure 20 Table 14. Figure 28 Investigation of different chromatographic columns.

[0267] Table 14 Column Analysis - Relative Retention Times

[0268]

[0269] The results showed that when the samples were detected using the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks ranged from 0.72% to 7.70%, indicating that the columns used in this method had good robustness.

[0270] 3.2.5.4.3.2 Stability Assessment

[0271] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 6h, 12h, and 24h. See Table 15.

[0272] Table 15 Stability Study - Retention Time

[0273]

[0274]

[0275] The results showed that the RSD of the retention time of the characteristic peak was between 0.02% and 0.33%, and the sample solution was stable within 24 hours.

[0276] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The aforementioned eight characteristic peaks will be included in subsequent investigations.

[0277] 3.2.6 Determination of characteristic peaks and establishment of reference spectra

[0278] 3.2.6.1 Verification results of 17 batches of standard decoction of kaempferia galanga

[0279] Using this method, characteristic spectral analysis was performed on 17 batches of samples, and relative retention times were calculated. See [link / reference]. Figures 29-30 Table 16. Figure 29 Characteristic chromatogram of standard decoction of Kaempferia galanga; Peak 5 (S): Cinnamic acid; Peak 6: 4-Methoxycinnamic acid; Peak 8: Ethyl p-methoxycinnamate; (S1-S8 from bottom to top are: SN-BT01, SN-BT02, SN-BT03, SN-BT04, SN-BT05, SN-BT06, SN-BT07, SN-BT08)

[0280] Figure 30 Characteristic spectrum of standard decoction of Kaempferia galanga; Peak 5 (S): Cinnamic acid; Peak 6: 4-methoxycinnamic acid; Peak 8: Ethyl p-methoxycinnamate (from bottom to top, S1-S9 are: SN-BT09, SN-BT10, SN-BT11, SN-BT12, SN-BT13, SN-BT14, SN-BT15, SN-BT16, SN-BT17).

[0281] Table 16 Relative Retention Time of Standard Decoction of Kaempferia galanga

[0282]

[0283] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peak values, eight peaks with good robustness were selected as characteristic peaks. According to the methodological investigation results and validation results from 17 batches of standard broth, the theoretical plate number, calculated based on the cinnamic acid peak, is tentatively set at no less than 5000.

[0284] 3.2.6.2 Establishment of Limits for Relative Retention Time

[0285] Table 17 summarizes the methodological examination items and validation results:

[0286] Table 17 Summary of RSD% for Methodological Results — Relative Retention Time / Retention Time

[0287]

[0288]

[0289] The relative retention times of each characteristic peak were stable, and the relative retention times of other test items were all within ±10% of the average value. The specified range of relative retention times for each peak was tentatively set at ±10%.

[0290] To better reflect the transfer of values, the relative retention times are consistent with those of the Kaempferia galanga granule formulation. The final specification stipulates that the test sample chromatogram should show 8 characteristic peaks, corresponding to the retention times of the 8 characteristic peaks in the reference chromatogram of the medicinal material. Peaks 5, 6, and 8 should correspond to the retention times of their respective reference peaks. The peak corresponding to the cinnamic acid reference peak is designated as peak S. The relative retention times of the remaining characteristic peaks and peak S are calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.26 (peak 1), 0.43 (peak 2), 0.65 (peak 3), 0.80 (peak 4), 1.14 (peak 7), and 1.28 (peak 8).

[0291] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize characteristic chromatograms of 10 batches of Kaempferia galanga standard decoction, and a reference chromatogram for the characteristic chromatograms of Kaempferia galanga standard decoction was established. See [link / reference]. Figure 31 . Figure 31 Compare with the characteristic chromatogram. Peak 5 (S): cinnamic acid; Peak 6: 4-methoxycinnamic acid; Peak 8: ethyl p-methoxycinnamate.

[0292] Example 4: UPLC Characteristic Chromatography of Kaempferia galanga Formulation Granules

[0293] 4.1. Experimental Instruments and Materials

[0294] High-performance liquid chromatographs: Agilent 1260 HPLC system; Waters e-2695 HPLC system; Thermo Fisher Vanquish Core HPLC system;

[0295] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0296] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0297] Ultrasonic cleaner: KQ600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0298] Chromatographic columns: Column 1 (Polar-Phenyl 120A, 4.6*250mm, 5μm); Column 2 (SVEATMC18Opal, 4.6*250mm, 5μm); Column 3 (Chromsil™ C18, 4.6*250mm, 5μm); Acetonitrile and phosphoric acid were chromatographic grade, water was ultrapure water, and all other reagents were analytical grade.

[0299] 4-Methoxycinnamic acid (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq22061001, purity 99.35%);

[0300] Cinnamic acid (China National Institutes for Food and Drug Control, batch number: 110786-202305, purity: 99.8%);

[0301] Ethyl p-methoxycinnamate (China National Institutes for Food and Drug Control, batch number: 110835-202005, purity: 99.4%);

[0302] Kaempferia galanga reference material (China National Institutes for Food and Drug Control, batch number: 121504-201203)

[0303] Kaempferia galanga formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers SN-KL01, SN-KL02, SN-KL03).

[0304] 4.2 Drafting Method

[0305] Chromatographic conditions and system suitability test

[0306] The column was packed with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5.0 μm particle size); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 270 nm. The theoretical plate number, calculated based on the cinnamic acid peak, should be no less than 5000.

[0307]

[0308] Preparation of the reference solution: Take 2g of *Kaempferia galanga* reference material, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 10ml of 50% methanol, shake well, filter, and use the filtrate as the reference solution. Separately, accurately weigh appropriate amounts of cinnamic acid and 4-methoxycinnamic acid reference standards, add methanol to prepare a mixed solution containing 25μg of cinnamic acid and 50μg of 4-methoxycinnamic acid per ml, as the reference solution. Also, accurately weigh an appropriate amount of ethyl p-methoxycinnamate reference standard, add methanol to prepare a solution containing 10μg of ethyl p-methoxycinnamate per ml, as the reference solution.

[0309] Preparation of the test solution: Take an appropriate amount of galangal formula granules, grind them finely, take 0.4g, place them in a stoppered conical flask, add 25ml of 50% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0310] The assay method involves precisely pipetting 10 μl each of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0311] 4.3 Chromatographic conditions and system suitability test

[0312] 4.3.1 Wavelength Selection

[0313] Based on the above-specified experimental conditions, a diode array detector was used to perform a full-band scan of the test solution, and chromatograms of the test solution were extracted at wavelengths of 230 nm, 250 nm, 270 nm, 280 nm, 290 nm, 310 nm, and 330 nm. See [link to chromatogram]. Figure 32 . Figure 32 Chromatograms of kaempferia galanga granules at different wavelengths.

[0314] The results showed that the chromatographic peak information was greater and the chromatographic baseline was more stable at a detection wavelength of 270 nm, so the detection wavelength was determined to be 270 nm.

[0315] 4.3.2 Selection of mobile phase

[0316] Based on the experimental conditions outlined above, the separation performance of three different mobile phases was investigated: acetonitrile-0.1% phosphoric acid, methanol-water, and methanol-0.1% phosphoric acid. (See attached figures.) Figure 33 . Figure 33 Mobile phase selection: The results showed that the chromatogram baseline was more stable and the chromatographic peaks were more numerous under the gradient elution condition of acetonitrile-0.1% phosphoric acid solution. Therefore, gradient elution of acetonitrile-0.1% phosphoric acid solution was selected as the mobile phase for the determination of the characteristic chromatogram of kaempferia galanga formulation granules.

[0317] 4.3.3 Column Temperature Investigation

[0318] Based on the above-established experimental conditions, the results were investigated at column temperatures of 25℃, 30℃, and 35℃. The results are shown below. Figure 34 . Figure 34 Column temperature investigation. The results showed that at a column temperature of 35℃, the chromatogram peaks were more symmetrical, the resolution was better, and the peaks were more complete. Therefore, the column temperature was determined to be 35℃.

[0319] 4.3.4 Flow velocity assessment

[0320] Based on the above-established experimental conditions, flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated. The results are shown below. Figure 35 . Figure 35 Flow rate determination. The results showed that a flow rate of 1.0 ml / min resulted in good peak shape and moderate resolution. Therefore, the flow rate was determined to be 1.0 ml / min.

[0321] 4.3.5 Delayedness Assessment

[0322] Based on the above-specified experimental conditions, a delay test was conducted. The results are shown below. Figure 36 . Figure 36 Delayed investigation.

[0323] The results showed that the sample had virtually no chromatographic peaks after 60 minutes, so the sample detection time was set at 60 minutes.

[0324] In summary, the chromatographic conditions and system suitability test for the characteristic chromatogram of Kaempferia galanga granules were determined as follows: Octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5.0 μm); acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 35℃; detection wavelength 270 nm. The theoretical plate number, calculated based on the cinnamic acid peak, should not be less than 5000.

[0325]

[0326] 4.4 Preparation of the test solution

[0327] 4.4.1 Examination of Extraction Methods

[0328] Take an appropriate amount of this product (batch number: SN-KL01), grind it into a fine powder, take 0.5g, place it in a stoppered conical flask, add 25ml of 50% methanol, seal tightly, and investigate the extraction methods for reflux and sonication respectively. The extraction time is 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the product. See Figure 37 . Figure 37Extraction methods were investigated. The results showed that there was little difference in the effectiveness of reflux and ultrasonic extraction of the test sample. The ultrasonic method was faster and simpler, so ultrasonic extraction was selected as the extraction method for the test sample.

[0329] 4.4.2 Investigation of Extraction Solvents

[0330] Take an appropriate amount of this product (batch number: SN-KL01), grind it into a fine powder, take 0.4g, place it in a stoppered conical flask, add 25ml each of methanol, 70% methanol, 50% methanol, and water, sonicate (600W power, 40kHz frequency) for 30 minutes, shake well, filter, and collect the filtrate to obtain the product. See Figure 38 . Figure 38 Extraction solvent investigation. The results showed that the extraction efficiency was high when the extraction solvent was 50% methanol, and the extraction solvent was tentatively set at 50% methanol.

[0331] 4.4.3 Investigation of Solvent Addition Amount

[0332] Take an appropriate amount of this product (batch number: SN-KL01), grind it into a fine powder, take 0.2g, place it in a stoppered conical flask, add 25ml, 50ml, and 100ml of 50% methanol respectively, sonicate (power 600W, frequency 40kHz) for 30 minutes, shake well, filter, and collect the filtrate to obtain the product. See Figure 39 . Figure 39 Solvent addition amount investigation. The results showed that when the extraction solvent addition amount was 25 ml, the peak shape and resolution of each chromatographic peak were good, and the peak size was appropriate. Therefore, the solvent volume was selected as 25 ml.

[0333] 4.4.4 Examination of extraction time

[0334] Take an appropriate amount of this product (batch number: SN-KL01), grind it into a fine powder, take 0.4g, place it in a stoppered conical flask, add 25ml of 50% methanol, and sonicate (power 600W, frequency 40kHz) for 15 minutes, 30 minutes, and 60 minutes respectively. Shake well, filter, and collect the filtrate to obtain the final product. See [link to product description] Figure 40 . Figure 40 Extraction time was examined.

[0335] The results showed that extraction was complete at a time of 30 minutes. Therefore, the extraction time was determined to be 30 minutes.

[0336] In summary, the preparation method of the galangal formula granule test solution is determined as follows: Take an appropriate amount of this product, grind it into a fine powder, take 0.4g, place it in a stoppered conical flask, add 25ml of 50% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, shake well, filter, and take the filtrate to obtain the product.

[0337] 4.5 Methodological Examination

[0338] 4.5.1 Chromatographic Peak Identification

[0339] Preparation of the test solution: Prepare the kaempferia galanga formula granule test solution according to the experimental conditions proposed above.

[0340] Preparation of reference solutions: Take 2g of Kaempferia galanga reference material, place it in a stoppered conical flask, add 50ml of water, decoct for 30 minutes, shake well, filter, evaporate to dryness, add 10ml of 50% methanol to dissolve, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of cinnamic acid, 4-methoxycinnamic acid, and ethyl p-methoxycinnamate reference standards, accurately weigh them, and add methanol to prepare mixed solutions containing 25μg of cinnamic acid, 50μg of 4-methoxycinnamic acid, and 10μg of ethyl p-methoxycinnamate per 1ml, respectively, as the reference solutions.

[0341] Preparation of negative control solution: Prepare negative control solution of Kaempferia galanga medicinal material according to the experimental conditions proposed above.

[0342] The characteristic peaks of the kaempferia galanga granule formulation were located. (See...) Figures 41-47 . Figure 41 For chromatographic peak identification; Figure 42 Spectrum of 4-methoxycinnamic acid - reference standard; Figure 43 Spectrum of 4-methoxycinnamic acid - Kaempferia galanga granules; Figure 44 Cinnamic acid spectrum - reference standard; Figure 45 Cinnamic acid spectrum - Kaempferia galanga granules; Figure 46 Spectrum of ethyl p-methoxycinnamate - reference standard; Figure 47 Spectrum of ethyl p-methoxycinnamate - Kaempferia galanga particles. Results showed that peak 5 (S) was cinnamic acid, peak 6 was 4-methoxycinnamic acid, and peak 8 was ethyl p-methoxycinnamate. The following methodological investigations examined eight characteristic peaks in the sample.

[0343] 4.5.2 Precision Test

[0344] Take the test solution of kaempferia galanga granules (batch number: SN-KL01), and inject it 6 times consecutively according to the proposed experimental method, 10 μl each time, and calculate the relative retention time of each characteristic peak. See Table 18.

[0345] Table 18 Precision Examination - Retention Time

[0346]

[0347]

[0348] The results showed that the retention time RSD of each characteristic peak of the sample ranged from 0.02% to 0.19%, indicating that the injection precision of this method was good.

[0349] 4.5.3 Repeatability Test

[0350] Six portions of galangal granules (batch number: SN-KL01) were prepared and tested according to the proposed experimental method. See Table 19.

[0351] Table 19 Repeatability Tests - Relative Retention Time

[0352]

[0353] The results showed that the relative retention time (RSD) of the six samples ranged from 0.00% to 1.96%, indicating that the method had good reproducibility.

[0354] 4.5.4 Intermediate Precision Examination

[0355] 4.5.4.1 Investigation with different instruments

[0356] Based on the above-planned experimental conditions, kaempferia galanga granules (batch number: SN-KL01) were weighed to prepare test solutions, which were then measured on Thermo Fisher Vanquish Core (instrument 1); Agilent 1260 (instrument 2); and Shimadzu Waterse2695 (instrument 3), respectively. See [link to relevant documentation]. Figure 48 Table 20. Figure 48 Different instruments were used for the investigation.

[0357] Table 20 Instrument Durability Test - Relative Retention Time

[0358]

[0359] The results showed that when the test samples were detected using the above three instruments, the RSD of the relative retention time of each characteristic peak was 0.72% to 6.93%, indicating that the method had good instrument durability.

[0360] 4.5.4.2 Investigations by different personnel and at different times

[0361] Based on the experimental conditions outlined above, different personnel (A and B) weighed kaempferia galanga granules (batch number: SN-KL01) at different times (T1 and T2) to prepare test samples for determination. See Table 21.

[0362] Table 21 Personnel and Time Assessment - Relative Retention Time

[0363]

[0364]

[0365] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0%-2.19%, indicating that the method had good intermediate precision.

[0366] 4.6 Durability Test

[0367] 4.6.1 Column robustness test

[0368] Based on the above-specified experimental conditions, analyses were conducted using chromatographic columns 1 (Polar-Phenyl 120A, 4.6*250mm, 5μm), 2 (SVEATM C18 Opal, 4.6*250mm, 5μm), and 3 (ChromsilTM C18, 4.6*250mm, 5μm), respectively. The results are shown in the table below. Figure 49 Table 22. Figure 49 Investigation of different chromatographic columns.

[0369] Table 22 Column robustness study - relative retention time

[0370]

[0371] The results showed that when the samples were detected using the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks ranged from 0.72% to 6.93%, indicating that the columns used in this method had good robustness.

[0372] 4.7 Stability Test

[0373] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 6h, 12h, and 24h. See Table 12-13.

[0374] Table 23 Stability Study - Retention Time

[0375]

[0376] The results showed that the RSD of the retention time of the characteristic peak was between 0.42% and 0.73%, indicating that the sample solution was stable within 24 hours.

[0377] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The above eight characteristic peaks will be included in subsequent investigations.

[0378] 4.8 Determination of characteristic peaks and establishment of reference spectra

[0379] 4.8.1 Validation results of three batches of kaempferia galanga granule formulation

[0380] Using this method, characteristic spectral analysis was performed on three batches of samples, and the relative retention times were calculated. See [link / reference]. Figure 50 Table 14.

[0381] Figure 50Characteristic spectrum of galangal formulation granules; Peak 5 (S): cinnamic acid; Peak 6: 4-methoxycinnamic acid; Peak 8: ethyl p-methoxycinnamate.

[0382] Table 24 Relative Retention Time of Kaempferia Granules

[0383]

[0384]

[0385] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peak values, a total of 8 peaks were selected as characteristic peaks. Based on the methodological investigation results and the particle validation results of 3 batches, the theoretical plate number, calculated using the cinnamic acid peak, is tentatively set to be no less than 5000.

[0386] 4.8.2 Establishment of Limits for Relative Retention Time

[0387] Table 25 summarizes the methodological examination items and validation results:

[0388] Table 25 Summary of RSD% for Methodological Results — Relative Retention Time / Retention Time

[0389]

[0390] The relative retention times of each characteristic peak were stable, and the relative retention times of all tested items were within ±10% of the average value. The specified range for the relative retention times of each peak is tentatively set at ±10%.

[0391] To enhance the adaptability of the method, the average relative retention time of different chromatographic columns was used as the final specified relative retention time, and the results are shown in Table 26.

[0392] Table 26 Average Relative Retention Time

[0393]

[0394] The final specification stipulates that the chromatogram of the test sample should show eight characteristic peaks, and the retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. Peaks 5, 6, and 8 should correspond to the retention times of their respective reference peaks. The peak corresponding to the cinnamic acid reference peak is designated as the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.25 (peak 1), 0.40 (peak 2), 0.62 (peak 3), 0.80 (peak 4), and 1.13 (peak 7).

[0395] Three batches of Kaempferia galanga granules were synthesized using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and a reference chromatogram of the characteristic chromatograms of Kaempferia galanga granules was established. (See...) Figure 51 . Figure 51 Compare with characteristic chromatograms; Peak 5 (S): Cinnamic acid; Peak 6: 4-Methoxycinnamic acid; Peak 8: Ethyl p-methoxycinnamic acid.

[0396] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing HPLC characteristic chromatogram of Radix Kaempferiae medicinal material decoction pieces, standard decoction and formula granules, comprising: A) dissolving and extracting the test sample raw material with a solvent to obtain a test solution; the test sample raw material is one or more of Radix Kaempferiae medicinal material, Radix Kaempferiae decoction pieces, Radix Kaempferiae standard decoction or Radix Kaempferiae formula granules; when the raw material is Radix Kaempferiae medicinal material and Radix Kaempferiae decoction pieces, step A) is to decoct Radix Kaempferiae medicinal material or Radix Kaempferiae decoction pieces with water, filter, dissolve the residue with 50% methanol, filter to obtain the test solution; when the raw material is Radix Kaempferiae standard decoction or Radix Kaempferiae formula granules, step A) is to ultrasonically treat Radix Kaempferiae standard decoction or Radix Kaempferiae formula granules with 50% methanol, cool, filter to obtain the test solution; B) determining the test solution by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of Radix Kaempferiae medicinal material decoction pieces, standard decoction and formula granules; preparing a reference solution: separately dissolving cinnamic acid, 4-methoxycinnamic acid and p-methoxycinnamic acid ethyl ester with methanol to obtain the reference solution; the high performance liquid chromatography conditions are: a C 18 column is used as the chromatographic column; the mobile phase A is acetonitrile solution and the mobile phase B is 0.1% phosphoric acid aqueous solution, gradient elution; the gradient elution is specifically: 0-10 min, A phase: 2%-10%, B phase: 98-90%; 10-30 min, A phase: 10%-25%, B phase: 90%-75%; 30-40 min, A phase: 25%-30%, B phase: 75%-70%; 40-50 min, A phase: 30%-45%, B phase: 70%-55%; 50-60 min, A phase: 45%-100%, B phase: 55%-0%; the detection wavelength is 270 nm. 2.The method of claim 1, wherein: the reference solution is determined by high performance liquid chromatography to obtain the chromatogram of the reference; and the components of Radix Kaempferiae medicinal material decoction pieces, standard decoction and formula granules are qualitatively determined according to the chromatogram of the reference.

3. The method of claim 2, wherein, The concentration of the reference solution is specifically: cinnamic acid is 25 μg / mL, 4-methoxycinnamic acid is 50 μg / mL, and p-methoxycinnamic acid ethyl ester is 10 μg / mL.

4. The method of claim 1, wherein, The specification of the chromatographic column is 250×4.6 mm 5 μm; the column temperature is 35 ℃; and the theoretical plate number calculated according to the cinnamic acid peak should not be less than 5000.

5. The method of claim 1, wherein, The flow rate of the mobile phase is 1.0 mL / min; and the injection amount is 10 μL.

6. The method of claim 1, wherein, The similarity of the HPLC characteristic chromatogram of Radix Kaempferiae medicinal material decoction pieces, standard decoction and formula granules is evaluated by a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain the HPLC standard characteristic chromatogram of Radix Kaempferiae medicinal material decoction pieces, standard decoction and formula granules composed of eight characteristic peaks, wherein peak 5 is cinnamic acid; peak 6 is 4-methoxycinnamic acid; and peak 8 is p-methoxycinnamic acid ethyl ester. In the characteristic spectrum of the Banlangen granules, with cinnamic acid as the reference peak S, the relative retention time of each characteristic peak to S is calculated, and the relative retention time is within ±10% of the specified value, and the specified value is 0.25 (peak 1), 0.40 (peak 2), 0.62 (peak 3), 0.80 (peak 4), 1.13 (peak 7), respectively; In the characteristic spectrum of the Banlangen standard decoction, with cinnamic acid as the reference peak S, the relative retention time of each characteristic peak to S is calculated, and the relative retention time is within ±10% of the specified value, and the specified value is 0.26 (peak 1), 0.43 (peak 2), 0.65 (peak 3), 0.80 (peak 4), 1.14 (peak 7), 1.28 (peak 8), respectively; In the characteristic spectrum of the Banlangen medicinal material or decoction piece, with cinnamic acid as the reference peak S, the relative retention time of each characteristic peak to S is calculated, and the relative retention time is within ±10% of the specified value, and the specified value is 0.27 (peak 1), 0.43 (peak 2), 0.65 (peak 3), 0.80 (peak 4), 1.14 (peak 7), 1.28 (peak 8), respectively.

7. The method of claim 1, wherein, The ratio of the mass g of the test sample raw material, the volume mL of water, and the volume mL of 50% methanol in step A) is 2:50:10; and the decoction time is 30 min.

8. The method of claim 1, wherein, The power of the ultrasonic is 600 W, and the frequency is 40 kHz; the ultrasonic time is 30 min. The ratio of the mass g of the test sample raw material and the volume mL of solvent is 0.4:

25.

9. A method for identifying the characteristic chromatogram of Radix Morae officinalis, standard decoction and formula granules, characterized in that, The detection is performed by the method of any one of claims 1-8, and the analysis detection result is obtained. The detection is performed by the method of any one of claims 1-8, and the analysis detection result is obtained.

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