Method for determining content of alkaloid substances in Jinqi blood glucose reducing tablets and application of method

Abstract

The invention provides a method for measuring the content of alkaloid substances in Jinqi blood glucose reducing tablets and application of the method, and relates to the technical field of medicine detection.The method comprises the steps that a test solution is taken, the peak area of a test sample is obtained through high performance liquid chromatography, and the content of the alkaloid substances in the test solution is calculated with the peak area of a berberine hydrochloride reference substance as a contrast; a mobile phase A of the high performance liquid chromatography is acetonitrile, a mobile phase B of the high performance liquid chromatography is an acid solution, and the acid solution comprises phosphoric acid with the mass concentration of 0.1%-0.2% and triethylamine with the mass concentration of 0.1%-0.2%; the alkaloid substances comprise epiberberine hydrochloride, coptisine hydrochloride, palmatine hydrochloride and berberine hydrochloride. The method can be used for simultaneously determining the contents of epiberberine hydrochloride, coptisine hydrochloride, palmatine hydrochloride and berberine hydrochloride in the Jinqi blood glucose reducing tablet, and the method is accurate and reliable and meets the requirements; the contents of epiberberine hydrochloride, coptisine hydrochloride, palmatine hydrochloride and berberine hydrochloride in the Jinqi blood glucose reducing tablet can be controlled by adopting a quantitative analysis of multi-components by single marker.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and in particular to a method for determining the content of alkaloids in Jinqi hypoglycemic tablets and its application. Background Technology

[0002] Currently, the commonly used method for detecting alkaloid content in Jinqi hypoglycemic preparations is the external standard method. This method uses high-performance liquid chromatography (HPLC) or ion mobility mass spectrometry (IM-MS) to employ a standard of known concentration as an external standard, and calculates the alkaloid content by measuring the peak area in the sample. Some studies have used ion mobility mass spectrometry to simultaneously determine eight components (including epiberberine, berberine, berberine, palmatine, etc.) in Jinqi hypoglycemic tablets. The results show that this method has good precision and repeatability, but it requires the separate preparation of single-reference solutions for each alkaloid, and the establishment of a separate standard curve for each component, making the operation cumbersome. In contrast, a single-analysis-multiple-evaluation method only requires a single labeled standard and only needs to establish a standard curve for the labeled standard, making the operation simpler. However, there is currently no single-analysis-multiple-evaluation method for Jinqi hypoglycemic preparations. Therefore, this invention is proposed. Summary of the Invention

[0003] One of the objectives of this invention is to provide a method for determining the content of alkaloids in Jinqi hypoglycemic tablets, thereby enabling multiple evaluations of the alkaloid content in Jinqi hypoglycemic tablets with a single measurement.

[0004] The second objective of this invention is to provide the application of the above-mentioned determination method in the quality control of Jinqi hypoglycemic tablets.

[0005] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for determining the content of alkaloids in Jinqi hypoglycemic tablets, comprising taking a test sample solution, obtaining the peak area of ​​the test sample by high performance liquid chromatography, using the peak area of ​​berberine hydrochloride as a reference, and calculating the content of alkaloids in the test sample solution; wherein the mobile phase A of the high performance liquid chromatography is acetonitrile, and the mobile phase B is an acid solution, wherein the acid solution comprises phosphoric acid with a mass concentration of 0.1%~0.2% and triethylamine with a mass concentration of 0.1%~0.2%; wherein the alkaloids include epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride.

[0006] Furthermore, the column packing material for the high-performance liquid chromatography is polar diethyl ether-linked phenyl-bonded silica gel.

[0007] Furthermore, the detection wavelength of the high-performance liquid chromatography is 345±2nm.

[0008] Furthermore, the column temperature for the high-performance liquid chromatography is 28~32℃.

[0009] Furthermore, the flow rate of the mobile phase is 0.9~1.1 ml / min.

[0010] Furthermore, the preparation method of the test solution includes taking Jinqi hypoglycemic tablets, adding methanol and ultrasonically extracting to obtain the test solution, wherein the amount of methanol used is 50-100 ml of methanol per 0.2 g of Jinqi hypoglycemic tablets.

[0011] Furthermore, the ultrasound duration is 30-60 minutes.

[0012] Furthermore, the conditions for the ultrasound include a frequency of 20 kHz and a power of 200~500 W.

[0013] Furthermore, the preparation method of the reference solution includes taking berberine hydrochloride and adding methanol to obtain a reference solution with a concentration of 100 μg / ml.

[0014] Secondly, this invention provides the application of the above-mentioned determination method in the quality control of Jinqi hypoglycemic tablets.

[0015] This invention provides a method for determining the content of alkaloids in Jinqi hypoglycemic tablets. The method can simultaneously determine the content of epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in Jinqi hypoglycemic tablets. The method has been validated for specificity, linearity, precision, intermediate precision, stability, repeatability, accuracy, and robustness, demonstrating that the method is accurate, reliable, and meets the requirements. Furthermore, results from multiple batches of samples show no significant difference between the content calculated using the external standard method and the one-test-multiple-evaluation method, indicating that the one-test-multiple-evaluation method can be used to control the content of epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in Jinqi hypoglycemic tablets. Attached Figure Description

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0017] Figure 1 Liquid chromatography of Jinqi hypoglycemic tablets under different chromatographic conditions provided by this invention Figure 1 ; Figure 2 Liquid chromatography of Jinqi hypoglycemic tablets under different chromatographic conditions provided by this invention Figure 2 ; Figure 3 Liquid chromatograms of Jinqi hypoglycemic tablets with different types of acids added to the mobile phase provided by the present invention; Figure 4 The liquid chromatogram of Jinqi hypoglycemic tablets with different concentrations of triethylamine added to the mobile phase provided by the present invention; Figure 5 The liquid chromatogram of the alkaloid reference solution provided for the specificity test of the present invention; wherein, A is epiberberine hydrochloride reference solution, B is berberine hydrochloride reference solution, C is palmatine hydrochloride reference solution, and D is berberine hydrochloride reference solution. Figure 6 The liquid chromatograms of the mixed reference solution, test solution, negative sample solution, and blank solvent provided for the specificity test of this invention; wherein, A is the mixed reference solution, B is the test solution, C is the Coptis chinensis negative sample solution, and D is the blank solvent; Figure 7 The alkaloid standard curves provided in Example 3 of the present invention are as follows: A is the standard curve of epiberberine hydrochloride, B is the standard curve of berberine hydrochloride, C is the standard curve of palmatine hydrochloride, and D is the standard curve of berberine hydrochloride. Detailed Implementation

[0018] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0019] In one aspect, this invention provides a method for determining the content of alkaloids in Jinqi hypoglycemic tablets, comprising taking a test sample solution, obtaining the peak area of ​​the test sample by high performance liquid chromatography (HPLC), using the peak area of ​​berberine hydrochloride as a reference, and calculating the content of alkaloids in the test sample solution; wherein the mobile phase A of the HPLC is acetonitrile, and the mobile phase B is an acid solution, wherein the acid solution comprises 0.1%~0.2% phosphoric acid and 0.1%~0.2% triethylamine by mass concentration; wherein the alkaloids include epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride.

[0020] This invention establishes a method for simultaneously determining the content of epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in Jinqi Jiangtang tablets. The method has been validated for specificity, linearity, precision, intermediate precision, stability, repeatability, accuracy, and robustness, demonstrating that the method is accurate, reliable, and meets the requirements. Furthermore, results from multiple batches of samples show no significant difference between the content calculated using the external standard method and the one-test-multiple-evaluation method, indicating that the one-test-multiple-evaluation method can be used to control the content of epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in Jinqi Jiangtang tablets.

[0021] Using berberine hydrochloride reference standard as a control, calculate the relative retention times of epiberberine hydrochloride, berberine hydrochloride, and palmatine hydrochloride. The relative retention times should be within 5% of the specified values. The specified values ​​for relative retention times and correction factors are shown in Table 1. Using the peak area of ​​berberine hydrochloride reference standard as a control, multiply by the correction factor to calculate the contents of epiberberine hydrochloride, berberine hydrochloride, and palmatine hydrochloride.

[0022] Table 1

[0023] In some specific embodiments, the acid solution comprises 0.1% phosphoric acid and 0.1% triethylamine by mass. In some specific embodiments, the chromatographic column packing for the high-performance liquid chromatography is polar diethyl ether-linked phenyl-bonded silica gel.

[0024] The ultraviolet full-wavelength spectra of epiberberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, and berberine hydrochloride reference standards all show that they have maximum absorption in the vicinity of 345 nm. In some specific embodiments, the detection wavelength of the high-performance liquid chromatography is 345 ± 2 nm.

[0025] In some specific embodiments, the column temperature of the high-performance liquid chromatography is 28~32℃, preferably 30℃. In some specific embodiments, the flow rate of the mobile phase is 0.9~1.1 ml / min, preferably 1.0 ml / min.

[0026] In some specific embodiments, the preparation method of the test solution includes taking Jinqi hypoglycemic tablets, adding methanol and ultrasonically extracting to obtain the test solution, wherein the amount of methanol used is 50-100 ml of methanol per 0.2 g of Jinqi hypoglycemic tablets, preferably 50 ml of methanol per 0.2 g of Jinqi hypoglycemic tablets.

[0027] In some specific embodiments, the ultrasound duration is 30-60 minutes, preferably 45 minutes. In some specific embodiments, the ultrasound conditions include a frequency of 20 kHz and a power of 200-500 W, preferably a frequency of 20 kHz and a power of 200 W.

[0028] In some specific embodiments, the preparation method of the reference solution includes taking berberine hydrochloride and adding methanol to obtain a reference solution with a concentration of 100 μg / ml.

[0029] According to another aspect of the present invention, the application of the above-described determination method in the quality control of Jinqi hypoglycemic tablets is also provided. The quality control of Jinqi hypoglycemic tablets includes using the above-described determination method to detect the content of alkaloids in Jinqi hypoglycemic tablets, thereby controlling the quality of Jinqi hypoglycemic tablets.

[0030] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0031] Experimental apparatus: Waters e2695 high-performance liquid chromatograph (HPLC), Waters Corporation, USA; SHIMADZU LC-20ADXR HPLC, Shimadzu Instruments Co., Ltd., Japan; KQ-400VDE dual-frequency digitally controlled ultrasonic instrument, Kunshan Ultrasonic Instrument Co., Ltd.; BAS224S-CW and SECUR125-1CN electronic balances, Sartorius GmbH, Germany; MS303 and AL204-IC electronic balances, Mettler Toledo GmbH, Germany. Column: Phenomenex Synergi Polar RP (4μm, 4.6×250mm), Welch UltimatePhenyl-Ether (5μm, 4.6×250mm), Silphila Megassil Polar Phenyl (5μm, 4.6×250mm), Agela Venusil XBP C18(L) (5μm, 4.6×250mm), SHIMADZU ShimNex CS C18 (5μm, 4.6×250mm), Agela Venusil MP C18 (5μm, 4.6×250mm), YMC-Pack ODS-AM (5μm, 4.6×250mm).

[0032] Reagents: Berberine hydrochloride reference standard (batch number: 110713-202015, content calculated as 85.9%), palmatine hydrochloride reference standard (batch number: 110732-201913, content calculated as 85.7%), and berberine hydrochloride reference standard (batch number: 112026-201802, content calculated as 94.0%) were purchased from the China National Institutes for Food and Drug Control. Epiberberine hydrochloride reference standard (batch number: 020007-202301, content calculated as 98.92%) was purchased from Shanghai Hongyong Biotechnology Co., Ltd.

[0033] Jinqi Hypoglycemic Tablets (Batch Nos.: 870090, 870106, 870107, 870128, 870129, 870103, 870244, 870245, 870247, 870236, 870239, 870095, 870133, 870138, 870142, 870124, 870121, 87) 0125, 870246, 870243, 870235); Honeysuckle ethanol extract (batch number: 870287); Astragalus extract (batch number: 870287); Pregelatinized starch (batch number: F2301003); Magnesium stearate (batch number: F0103); Microcrystalline cellulose (batch number: F221112); All samples were provided by Longshunrong Pharmaceutical Factory. Acetonitrile was chromatographically pure; water was ultrapure water; methanol, anhydrous ethanol, phosphoric acid, glacial acetic acid, formic acid, and triethylamine were all analytically pure.

[0034] Take 20 tablets of Jinqi hypoglycemic tablets (batch number 870090), remove the coating, accurately weigh each tablet to an average weight of 0.518535g, grind them into a fine powder, and use them as test samples for Examples 1 to 3.

[0035] Example 1: Comparison of different chromatographic conditions 1.1 Test solution: Accurately weigh 0.2g of the test sample and place it in a stoppered conical flask. Add 50ml of 50% ethanol solution, weigh the sample, and sonicate (power 200W, frequency 40KHz) for 60 minutes. After cooling, weigh the sample again. Make up the weight loss with 50% ethanol solution, shake well, filter, and collect the filtrate.

[0036] 1.2 Comparison of different mobile phases and chromatographic columns The "1.1 Test Solution" was injected and analyzed under different chromatographic conditions. The mobile phase conditions and resolution results are shown in Table 2, and the chromatograms are shown in the figure. Figure 1 and Figure 2 As shown, peak 1 is epiberberine hydrochloride, peak 2 is berberine hydrochloride, peak 3 is palmatine hydrochloride, and peak 4 is berberine hydrochloride.

[0037] Table 2

[0038] The results showed that the resolution of each component on the column packed with polar diethyl ether-linked phenyl-bonded silica gel (Phenomenex Synergi Polar RP) was significantly better than that on the column packed with octadecylsilane-bonded silica gel. When the mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, the chromatographic peaks of each component exhibited severe tailing. Adding buffer salt solution or ion-pairing reagents to the mobile phase caused significant damage to the column. When the mobile phase was acetonitrile-0.1% phosphoric acid solution (containing 0.1% triethylamine), the chromatographic peaks of each component showed good resolution, and the purity angles of each peak were all within acceptable limits. Therefore, condition 5 was the optimal chromatographic condition.

[0039] 1.3 Comparison of adding different types of acids to the mobile phase 0.1% glacial acetic acid, 0.1% formic acid, and 0.1% phosphoric acid were added to the aqueous mobile phase, respectively. The Jinqi hypoglycemic tablets sample solution was analyzed under condition 5, and the changes in each chromatographic peak were compared. The chromatogram is shown below. Figure 3 As shown, the chromatographic peak shape was slightly worse under the conditions of 0.1% glacial acetic acid and 0.1% formic acid. Therefore, adding phosphoric acid to the aqueous phase of the mobile phase yielded the best results.

[0040] 1.4 Comparison of different concentrations of triethylamine added to the mobile phase Compare the changes in chromatographic peaks when using 0.1% phosphoric acid aqueous solution containing different concentrations of triethylamine (0.05%, 0.1%, 0.15%, and 0.2%) as the mobile phase. Figure 4 As shown, when the triethylamine concentration is 0.05%, the peak shape of berberine hydrochloride is slightly poor. When the triethylamine concentration is 0.1%, 0.15%, and 0.2%, the peak shapes of each chromatographic peak are good. It can be seen that adding 0.1% triethylamine can improve the peak shape of each chromatographic peak.

[0041] 1.5 The preferred chromatographic conditions are as follows: a polar ether-linked phenyl-bonded silica gel column (Phenomenex Synergi Polar RP, 4µm, 4.6×250mm) as the stationary phase; acetonitrile-0.1% phosphoric acid solution (containing 0.1% triethylamine) (23:77) as the mobile phase; a detection wavelength of 345nm; a column temperature of 30℃; an injection volume of 5µl; and a flow rate of 1.0ml per minute. The following examples all use these chromatographic conditions for determination.

[0042] Example 2 Comparison of different test sample solution preparation methods 2.1 Test solutions prepared with different extraction solvents Accurately weigh 0.2 g of the test sample powder and place it in a stoppered conical flask. Add 50 ml of the extraction solvent shown in Table 3, weigh the flask, and sonicate (40 kHz, 200 W) for 1 hour. Perform two replicates for each solvent. After cooling, weigh the flask again and replenish the lost weight with the respective solvent. Shake well and filter to obtain the final product. The results are shown in Table 3, in mg / tablet. Based on the content of the target compounds, the extracting solvent, methanol, yielded higher contents of epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride.

[0043] Table 3

[0044] 2.2 Test solutions prepared by different extraction methods Accurately weigh 0.2 g of the sample powder and place it in a stoppered conical flask. Add 50 ml of methanol and weigh again. Extract the sample by sonication (40 kHz, 200 W), reflux (80 °C), and shaking (120 Hz) for 1 hour, respectively. Perform two replicates for each extraction method. After cooling, weigh again and replenish the lost weight with methanol. Shake well and filter to obtain the final product. The results are shown in Table 4 (unit: mg / tablet). Ultrasonic extraction is considered more suitable.

[0045] Table 4

[0046] 2.3 Test solutions prepared at different extraction times Accurately weigh 0.2 g of the test sample powder, add 50 ml of methanol, weigh again, and ultrasonically extract (40 kHz, 200 W) for 15 min, 30 min, 45 min, and 60 min respectively. Perform duplicate extractions for each extraction time. After cooling, weigh again, replenish the lost weight with methanol, shake well, and filter. The results are shown in Table 5. Under different extraction times, the contents of epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride were relatively high after 45 min of extraction.

[0047] Table 5

[0048] 2.4 Test solutions prepared with different solvent volumes Accurately weigh 0.2 g of the test sample powder, and perform six parallel aliquots. Add 50 ml, 75 ml, and 100 ml of methanol respectively, weigh the samples, and perform ultrasonic extraction (40 kHz, 200 W) for 45 min. Perform two parallel aliquots for each solvent volume, cool the samples, weigh them again, and replenish the lost weight with methanol. Shake well, filter, and obtain the final product. The results are shown in Table 6. Under different solvent volumes, the highest contents of the target compounds epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride were found when the solvent volume was 50 ml.

[0049] Table 6

[0050] 2.5 Test solutions prepared with different ultrasonic frequencies and ultrasonic powers Accurately weigh 0.2 g of the test sample powder, make eight parallel portions, add 50 ml of methanol to each portion, weigh, and ultrasonically extract for 45 min. The ultrasonic parameters are shown in Table 6. For each extraction parameter, make two parallel portions, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and obtain the final product. The determination results are shown in Table 7. When the ultrasonic extraction parameters are 20 kHz and 200 W, the contents of the target compounds epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride are the highest.

[0051] Table 7

[0052] 2.6 Preferred preparation method of the test solution: Take 0.2g of the test sample powder, weigh it accurately, add 50ml of methanol accurately, weigh it, sonicate (20kHz, 200W) for 45min, cool it, weigh it again, make up the weight loss with methanol, shake it well, filter it, and the solution is obtained.

[0053] Example 3 Methodological Validation 3.1 Solution Preparation 3.1.1 Preparation of test solution: Prepared according to “2.6 Preferred method for preparing test solution”.

[0054] 3.1.2 Preparation of mixed reference solution: Accurately weigh epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride reference standards, respectively, and add methanol to prepare a mixed solution containing 6 μg of epiberberine hydrochloride, 30 μg of berberine hydrochloride, 12 μg of palmatine hydrochloride, and 100 μg of berberine hydrochloride per 1 ml.

[0055] 3.1.3 Preparation of negative sample solution: Weigh 12.5g of Astragalus membranaceus alcohol extract and 23.7g of Lonicera japonica water extract, mix and dry under reduced pressure (80℃) to obtain a dry paste, pulverize into a fine powder, add 6.7g of microcrystalline cellulose, 3.9g of pregelatinized starch and 0.2g of magnesium stearate, mix well to obtain Coptis chinensis negative sample powder. Prepare Coptis chinensis negative sample solution according to "2.6 Preferred method for preparing test solution".

[0056] 3.2 Chromatographic conditions: The determination was carried out according to “1.5 Preferred chromatographic conditions”.

[0057] 3.3 Specificity Test: Mixed reference solution, test solution, and negative sample solution were prepared according to methods “3.1.1” to “3.1.3”, and analyzed under the chromatographic conditions described in “3.2”. The theoretical plate number and resolution are shown in Table 8, and the chromatograms are shown below. Figure 5 and Figure 6 As shown.

[0058] Table 8

[0059] The results showed that the test sample chromatogram and the mixed reference standard chromatogram had corresponding chromatographic peaks at the same retention times, and the peak shapes were good. The negative sample chromatogram showed no chromatographic peaks at the same retention times, indicating that this method has good specificity and no interference from negative samples.

[0060] 3.4 System Applicability The mixed reference solution was prepared according to method "3.1.2" and analyzed under the chromatographic conditions described in "3.2". The resolution of the five-injection mixed reference solution was summarized, and the RSD of the peak areas of epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in the mixed reference solution was calculated. The results are shown in Table 9. The theoretical plate number is shown in Table 10.

[0061] Table 9

[0062] The results showed that after five consecutive injections, the RSD of the peak areas of epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in the mixed reference solution was less than 2%, which met the requirements of "0512 High Performance Liquid Chromatography" in Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0063] 3.5 Repeatability Test Six test solutions were prepared according to method “3.1.1”, and analyzed according to the chromatographic conditions “3.2”. The results are shown in Table 10.

[0064] Table 10

[0065] Table 10 shows that the contents and relative standard deviations (RSD%) of epiberberine hydrochloride, berberine hydrochloride, and palmatine hydrochloride all meet the precision requirements (RSD < 3%) for the analyte content level of 1 mg / g in the "9101 Analytical Method Validation Guidelines" of the 2020 edition of the Chinese Pharmacopoeia, Part IV. The relative standard deviation (RSD%) of berberine hydrochloride meets the precision requirements (RSD < 2%) for the analyte content level of 10 mg / g in the "9101 Analytical Method Validation Guidelines" of the 2020 edition of the Chinese Pharmacopoeia, Part IV.

[0066] 3.6 Accuracy Test Accurately weigh 0.1 g of the test sample powder and accurately add it to 50 ml of the mixed reference solution (containing 3.02497 μg / ml epiberberine hydrochloride, 14.8802 μg / ml berberine hydrochloride, 6.04355 μg / ml palmatine hydrochloride, and 49.4097 μg / ml berberine hydrochloride, dissolved in methanol). Prepare the test solution according to method “3.1.1”. Accurately pipette 5 μl each of the mixed reference solution and the test solution prepared according to method “3.1.2”, and analyze them according to the chromatographic conditions in “3.2”. The results are shown in Table 11.

[0067] Table 11

[0068] Table 11 shows that the average recoveries and RSDs of epiberberine hydrochloride, berberine hydrochloride, and palmatine hydrochloride all meet the accuracy requirements (recovery limits 90-108%) for the analyte concentration level of 1 mg / g as outlined in the "9101 Analytical Method Validation Guidelines" of the 2020 edition of the Chinese Pharmacopoeia, Part IV. The average recoveries and RSDs of berberine hydrochloride also meet the accuracy requirements (recovery limits 92-105%) for the analyte concentration level of 10 mg / g as outlined in the "9101 Analytical Method Validation Guidelines" of the 2020 edition of the Chinese Pharmacopoeia, Part IV.

[0069] 3.7 Preparation of Standard Curve Accurately weigh appropriate amounts of epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride reference standards, and prepare mixed reference solutions with concentrations shown in Table 12 using methanol. Accurately inject 5 μl of each solution into the liquid chromatograph and determine the peak area under the chromatographic conditions described above. The results are shown in Table 12. Perform linear regression with reference standard concentration as the x-axis (μg / ml) and peak area as the y-axis, as shown in Table 12. Figure 7 As shown.

[0070] Table 12

[0071] The results showed that the concentrations of epiberberine hydrochloride and its peak area exhibited good linearity in the range of 1.2100–12.0999 μg / ml; the concentrations of berberine hydrochloride and its peak area exhibited good linearity in the range of 5.9521–59.5208 μg / ml; the concentrations of palmatine hydrochloride and its peak area exhibited good linearity in the range of 2.4174–24.1743 μg / ml; and the concentrations of berberine hydrochloride and its peak area exhibited good linearity in the range of 19.7693–197.6387 μg / ml.

[0072] 3.8 Intermediate Precision Test The test sample solution was prepared by different analysts on different instruments according to the method in "3.1.1", and analyzed according to the chromatographic conditions in "3.2". The results are shown in Table 13, with units of mg / tablet.

[0073] Table 13

[0074] The results showed that the relative standard deviations (RSD%) of the results for epiberberine hydrochloride, berberine hydrochloride, and palmatine hydrochloride obtained by different analysts on different instruments were all within the range of 0.6% to 0.8%, which meets the precision requirements (RSD < 3%) for the content level of the analyte at 1 mg / g as specified in the "9101 Analytical Method Validation Guidelines" of the 2020 edition of the Chinese Pharmacopoeia, Part IV. The relative standard deviation (RSD%) of the results for berberine hydrochloride obtained by different analysts on different instruments was 0.9%, which meets the precision requirements (RSD < 2%) for the content level of the analyte at 10 mg / g as specified in the "9101 Analytical Method Validation Guidelines" of the 2020 edition of the Chinese Pharmacopoeia, Part IV.

[0075] 3.9 Durability Test 3.9.1 Stability Test Prepare one test solution according to the method in “3.1.1”. Accurately pipette 5 μl of the test solution and perform the determination at 0, 2, 6, 10, 18, 24 and 36 hours according to the chromatographic conditions in “3.2”. The determination results are shown in Table 14.

[0076] Table 14 Stability test results (n=7)

[0077] The results showed that the contents of epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride in the test solution at different time points and the RAD value at 0 hours were all less than 1%, indicating that the test solution had good stability within 36 hours.

[0078] 3.9.2 Column Temperature Durability Prepare the test solution according to the method in "3.1.1". Perform the determination according to the chromatographic conditions in "3.2", with column temperatures of 28℃, 30℃ and 32℃, and the results are shown in Table 15.

[0079] Table 15 Column Temperature Durability Test Results (n=3)

[0080] The results showed that the RAD values ​​of the content results of epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride at column temperatures of 30±2℃ and 30℃ were all less than 2%, indicating that the method has good durability in the column temperature range of 28~32℃.

[0081] 3.9.3 Flow rate durability Prepare the test solution according to the method in "3.1.1". Perform the determination according to the chromatographic conditions in "3.2", with column temperatures of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min. The results are shown in Table 16.

[0082] Table 16 Results of flow velocity durability tests (n=3)

[0083] The results showed that the RAD values ​​of the contents of epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride under different flow rates of 0.9 and 1.1 ml / min and the contents under 1.0 ml / min were all less than 2%, indicating that the method has good flow rate robustness.

[0084] 3.9.4 Detection Wavelength Robustness Prepare the test solution according to the method in "3.1.1". Perform the determination at wavelengths of 340 nm and 350 nm under the chromatographic conditions in "3.2", and the results are shown in Table 17.

[0085] Table 17 Wavelength durability test results; Flow rate durability test results (n=3)

[0086] The results showed that the RAD values ​​of the content results of epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride at the detection wavelength of 345±5nm and the content results at the detection wavelength of 345nm were all less than 2%, indicating that the method has good wavelength robustness.

[0087] Example 4 Sample Determination Take appropriate amounts of different batches of Jinqi hypoglycemic tablets and prepare test solutions according to the above-described method. Perform the determination under the above chromatographic conditions, and the results are shown in Table 18.

[0088] Table 18. Content determination results of multiple batches of samples (mg / tablet)

[0089] Example 5: Examination of Correction Factors 5.1 Calculation of Correction Factor According to the formula f=(CA / AA) / (CS / AS) (where CA is the concentration of the analyte, AA is the peak area of ​​the analyte, CS is the concentration of the reference substance, and AS is the peak area of ​​the reference substance), the calculation of correction factors commonly employs multi-point correction and averaging. However, when one or two points have significant deviations (especially the first and last points), they can affect the overall average, leading to a certain deviation in the calculated value. Calculating the correction factor using the ratio of the slopes of the standard curve is more scientific and reasonable, as even if several points have significant deviations, their impact on the overall slope of the standard curve is minimal. Furthermore, slope correction eliminates the need to calculate the correction factor for each concentration and then average it; it can be directly calculated using the slope of the standard curve, which is relatively faster. Therefore, in this experiment, using berberine hydrochloride as the reference substance, the slope method was used to calculate the correction factors for epiberberine hydrochloride, berberine hydrochloride, and palmatine hydrochloride based on the measurement results in "3.7," resulting in 1.2537, 1.1051, and 0.9397, respectively.

[0090] 5.2 Reproducibility test of the correction factor 5.2.1 Column Temperature Investigation With other chromatographic conditions unchanged, the column temperature was set to 28℃, 30℃, and 32℃ respectively. The mixed reference solution under section "3.1.2" was injected, the peak area was recorded, and the correction factor was calculated. The results are shown in Table 19. At different column temperatures, the RSD values ​​of the correction factors between berberine hydrochloride and each component were all less than 5%, indicating good reproducibility.

[0091] Table 19 Correction factor values ​​for measurements at different column temperatures (n=3)

[0092] 5.2.2 Flow velocity investigation With other chromatographic conditions unchanged, flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min were set, and the mixed reference solution under section "3.1.2" was injected. Peak areas were recorded, and correction factors were calculated. The results are shown in Table 20. At different flow rates, the RSD values ​​of the correction factors between berberine hydrochloride and each component were all less than 5%, indicating good reproducibility.

[0093] Table 20 Correction factor values ​​for measurements at different flow rates (n=3)

[0094] 5.2.3 Investigation of Detection Wavelength With other chromatographic conditions unchanged, detection wavelengths of 340 nm, 343 nm, 345 nm, 347 nm, and 350 nm were set, and the mixed reference solution under section "3.1.2" was injected. Peak areas were recorded, and correction factors were calculated. The results are shown in Table 21. At a detection wavelength of 345±5 nm, the RSD value of the correction factor between berberine hydrochloride and berberine hydrochloride was greater than 5%, while at a detection wavelength of 345±2 nm, the RSD values ​​of the correction factors between berberine hydrochloride and each component were all less than 5%, indicating that the correction factors between berberine hydrochloride and each component have good robustness in the detection wavelength range of 345±2 nm.

[0095] Table 21 Correction factor values ​​for measurements at different detection wavelengths (n=5)

[0096] 5.2.4 Column Investigation Under constant chromatographic conditions, the mixed reference solution described in section "3.1.2" was injected into a Waters e2695 and a SHIMADZU LC-20ADXR high-performance liquid chromatograph using Phenomenex Synergi Polar RP (4 μm, 4.6 × 250 mm), Welch Ultimate Phenyl-Ether (5 μm, 4.6 × 250 mm), Siphila Megassil Polar Phenyl (5 μm, 4.6 × 250 mm), Agela Venusil MP C18 (5 μm, 4.6 × 250 mm), and YMC-Pack ODS-AM (5 μm, 4.6 × 250 mm) columns, respectively. Peak areas were recorded, and correction factors were calculated. The results are shown in Tables 22 and 23. Under different instruments and columns, the RSD values ​​of the correction factors between berberine hydrochloride and each component were all less than 5%, indicating good reproducibility.

[0097] Table 22 Correction factor values ​​for measurements using different instruments and chromatographic columns with different packing materials (n=3)

[0098] Phenomenex is a phenyl-bonded silica gel with polar diethyl ether as a filler; YMC and Agela are octadecylsilane-bonded silica gels as fillers.

[0099] Table 23 Correction factor values ​​for measurements using different instruments and the same packing column (n=3)

[0100] 5.2.5 Chromatographic peak localization of the analyte Referring to the chromatographic localization method, relative retention time (the ratio of the retention time of the analyte to that of the reference) was used as the localization standard. This parameter was investigated using different brands of chromatographic columns on different brands of instruments (the columns described in "5.2.4" were selected). The results are shown in Tables 24 and 25. On columns with different packing materials, the relative retention times of each component fluctuated significantly (RSD > 5%), while on columns with the same packing material, the relative retention times of each component fluctuated less significantly (RSD < 5%). This indicates that relative retention time should be used for target peak localization when the chromatographic column packing material is fixed.

[0101] Table 24 Relative retention time values ​​determined by different instruments and different packing columns (n=3)

[0102] Table 25 Relative retention time values ​​determined by different instruments and chromatographic columns with the same packing material (n=3)

[0103] 5.2.6 Comparison of Single Measurement with Multiple Evaluation Method and External Standard Method Data from multiple batches of samples were analyzed, and the content of each component was calculated using the one-test-multiple-evaluation method. The results were then compared with those calculated using the external standard method. The results are shown in Table 26. The RAD values ​​calculated by both methods were less than 5%, and there was no significant difference between the two methods. Therefore, the correction factor in this experiment is accurate and reliable.

[0104] Table 26 Comparison of the content of each component calculated using the one-test-multiple-evaluation method and the external standard method (mg / tablet)

[0105] Note: a-external standard method, b-one-test-multiple-evaluation method.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for determining the content of alkaloids in Jinqi hypoglycemic tablets, characterized in that, The process includes taking the test sample solution, obtaining the peak area of ​​the test sample by high performance liquid chromatography, using the peak area of ​​berberine hydrochloride reference standard as a reference, and calculating the content of alkaloids in the test sample solution; The mobile phase A of the high-performance liquid chromatography is acetonitrile, and the mobile phase B is an acid solution, wherein the acid solution comprises phosphoric acid with a mass concentration of 0.1% to 0.2% and triethylamine with a mass concentration of 0.1% to 0.2%. The alkaloids include epiberberine hydrochloride, berberine hydrochloride, palmatine hydrochloride, and berberine hydrochloride.

2. The determination method according to claim 1, characterized in that, The column packing material for the high-performance liquid chromatography method is polar diethyl ether-linked phenyl-bonded silica gel.

3. The determination method according to claim 1, characterized in that, The detection wavelength of the high-performance liquid chromatography method is 345±2nm.

4. The determination method according to claim 1, characterized in that, The column temperature for the high-performance liquid chromatography method is 28~32℃.

5. The determination method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.9~1.1 ml / min.

6. The determination method according to claim 1, characterized in that, The preparation method of the test solution includes taking Jinqi hypoglycemic tablets, adding methanol and ultrasonically extracting to obtain the test solution, wherein the amount of methanol used is 50-100 ml of methanol per 0.2 g of Jinqi hypoglycemic tablets.

7. The determination method according to claim 6, characterized in that, The ultrasound session lasted 30 to 60 minutes.

8. The determination method according to claim 6, characterized in that, The conditions for the ultrasound include a frequency of 20kHz and a power of 200~500W.

9. The determination method according to claim 1, characterized in that, The preparation method of the reference solution includes taking berberine hydrochloride and adding methanol to obtain a reference solution with a concentration of 100 μg / ml.

10. The application of the determination method according to any one of claims 1-9 in the quality control of Jinqi hypoglycemic tablets.

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