A method for simultaneously detecting contents of catalpol, rehmannioside D and chlorogenic acid in medicinal liquid

Abstract

The application relates to the technical field of pharmaceutical analysis, and particularly discloses a detection method for simultaneously detecting the contents of catalpol, rehderin D and chlorogenic acid in medicinal liquid. The detection method for simultaneously detecting the contents of catalpol, rehderin D and chlorogenic acid in medicinal liquid adopts high performance liquid chromatography detection; the gradient elution program is as follows: 0-35 min, 1% of methanol and 99% of 0.18wt% phosphoric acid solution; 35-45 min, the methanol is increased from 1% to 5%, and the 0.18wt% phosphoric acid solution is decreased from 99% to 95%; 45-60 min, the water is increased from 0% to 20%, the methanol is increased from 5% to 30%, and the 0.18wt% phosphoric acid solution is decreased from 95% to 50%; 60-90 min, the water is decreased from 20% to 0%, the methanol is increased from 30% to 50%, and the 0.18wt% phosphoric acid solution is 50%. The application can accurately detect the contents of catalpol, rehderin D and chlorogenic acid in medicinal liquid, and control the quality of the medicinal liquid.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for simultaneously detecting the contents of catalpol, rehmannia glycoside D, and chlorogenic acid in a pharmaceutical solution. Background Technology

[0002] Traditional Chinese medicine (TCM) is a treasure of the Chinese nation, with its history dating back over 5,000 years to Shennong's tasting of hundreds of herbs. TCM herbs originate from nature and are generally produced in a wide range of areas. Due to differences in growing environments, cultivation methods, harvesting methods, and seasons, the quality of medicinal materials varies, and the content of their active ingredients differs. In recent years, with the further scientific development of TCM, various methods for detecting the medicinal properties and potency of TCM ingredients have been gradually developed.

[0003] A traditional Chinese medicine liquid is prepared from herbs such as Polygonum multiflorum vine, Spatholobus suberectus, Rehmannia glutinosa, Prunus mume, Zanthoxylum bungeanum, Cnidium monnieri, Kochia scoparia, Tribulus terrestris, Dictamnus dasycarpus, and Phellodendron chinense. The main active ingredients in this liquid are catalpol, rehmannia glycoside D, and chlorogenic acid. To test the quality of this liquid, it is necessary to detect the content of these three main active ingredients. Current technology generally uses high-performance liquid chromatography (HPLC) to detect the content of catalpol, rehmannia glycoside D, and chlorogenic acid separately. However, this method is time-consuming and labor-intensive, and there is no existing method for simultaneously detecting the content of all three ingredients. Therefore, there is an urgent need for a method that can simultaneously detect the content of catalpol, rehmannia glycoside D, and chlorogenic acid in the liquid. Summary of the Invention

[0004] This application provides a method for precisely detecting the content of catalpol, rehmannia glycoside D, and chlorogenic acid in a pharmaceutical solution.

[0005] The method for simultaneously detecting the contents of catalpol, rehmannia glycoside D, and chlorogenic acid in a drug solution provided in this application adopts the following technical solution:

[0006] A method for simultaneously detecting the contents of catalpol, rehmannia glycoside D, and chlorogenic acid in a medicinal solution, wherein the medicinal solution comprises ten medicinal herbs decocted together: Polygonum multiflorum vine, Spatholobus suberectus, Rehmannia glutinosa, Prunus mume, Zanthoxylum bungeanum, Cnidium monnieri, Kochia scoparia, Tribulus terrestris, Dictamnus dasycarpus, and Phellodendron chinense. The detection method comprises the following steps: preparation of the test solution, preparation of the mixed reference solution, and detection by high performance liquid chromatography.

[0007] The mobile phase in the high-performance liquid chromatography detection conditions includes water, methanol, and 0.18 wt% phosphoric acid solution;

[0008] The high-performance liquid chromatography method employs a gradient elution procedure;

[0009] The gradient elution procedure is as follows:

[0010] From 0 min to 35 min, methanol was 1% and 0.18 wt% phosphoric acid solution was 99%.

[0011] Between 35 and 45 minutes, the methanol concentration increased from 1% to 5%, and the 0.18 wt% phosphoric acid solution decreased from 99% to 95%.

[0012] From 45 to 60 minutes, the water content increased from 0% to 20%, the methanol content increased from 5% to 30%, and the 0.18 wt% phosphoric acid solution decreased from 95% to 50%. From 60 to 90 minutes, the water content decreased from 20% to 0%, the methanol content increased from 30% to 50%, and the 0.18 wt% phosphoric acid solution remained at 50%.

[0013] In the above technical solution, through a specific elution procedure, from 0 min to 35 min, methanol is 1% and 0.18 wt% phosphoric acid solution is 99%; from 35 min to 45 min, methanol increases from 1% to 5% and 0.18 wt% phosphoric acid solution decreases from 99% to 95%; from 45 min to 60 min, water increases from 0% to 20%, methanol increases from 5% to 30%, and 0.18 wt% phosphoric acid solution decreases from 95% to 50%; from 60 min to 90 min, water decreases from 20% to 0%, methanol increases from 30% to 50%, and 0.18 wt% phosphoric acid solution is 50%, thus achieving specific colorimetric... The spectrum simultaneously displays the characteristic peaks of catalpol, rehmannia glycoside D, and chlorogenic acid. This not only eliminates interference from other impurities on the characteristic peaks of catalpol, rehmannia glycoside D, and chlorogenic acid, but also eliminates mutual interference among the characteristic peaks of catalpol, rehmannia glycoside D, and chlorogenic acid, ensuring the integrity of the characteristic peaks of catalpol, rehmannia glycoside D, and chlorogenic acid. It allows for better differentiation of the characteristic peaks of catalpol, rehmannia glycoside D, and chlorogenic acid, and enables precise and accurate detection of the content of catalpol, rehmannia glycoside D, and chlorogenic acid in the drug solution based on the peak area. Optimizing the detection of the content of catalpol, rehmannia glycoside D, and chlorogenic acid in the drug solution is beneficial for better control of the drug solution quality and further improvement of the drug solution quality.

[0014] Preferably, the detection wavelength in the high-performance liquid chromatography detection conditions is 210 nm.

[0015] In the above technical solution, by specifically selecting a detection wavelength of 210nm, the accuracy of the detection results can be further improved.

[0016] Preferably, the flow rate in the high performance liquid chromatography detection conditions is 0.6 mL / min.

[0017] In the above technical solution, by specifically selecting a flow rate of 0.6 mL / min, it is possible to further and fully separate catalpol, rehmannia glycoside D, and chlorogenic acid in the drug solution, thereby further improving the accuracy of the detection results.

[0018] Preferably, the injection volume in the high performance liquid chromatography detection conditions is 15–20 μL.

[0019] In the above technical solution, by specifically selecting an injection volume of 15–20 μL, the mutual interference between characteristic peaks is further reduced, and the accuracy of the detection results is further improved.

[0020] Preferably, the test solution is a drug solution diluted with methanol, and the volume ratio of the drug solution to methanol is (1 mL to 2 mL): (18 mL to 19 mL).

[0021] In the above technical solution, the test solution is prepared by mixing the drug solution and methanol. The volume ratio of the drug solution to methanol is (1mL~2mL):(18mL~19mL), which can better promote the separation of the characteristic peaks of catalpol, rehmannia glycoside D and chlorogenic acid and further reduce the interference of the characteristic peaks of other impurities, thereby further improving the accuracy of the detection results.

[0022] Preferably, the preparation method of the mixed reference solution for high performance liquid chromatography is as follows: accurately weigh appropriate amounts of catalpol reference standard, rehmannia glutinosa D reference standard, and chlorogenic acid reference standard and mix them to obtain a mixture; add an appropriate amount of diluent to the mixture to prepare the mixed reference solution.

[0023] The diluent was prepared by mixing methanol and 0.18 wt% phosphoric acid solution in a volume ratio of 1:99.

[0024] In the above technical solution, by preparing the reference standard using a mixture of catalpol, rehmannia glycoside D, and chlorogenic acid reference standards, the chromatograms of catalpol, rehmannia glycoside D, and chlorogenic acid reference standards can be accurately detected. By comparing and calculating the peak areas of the characteristic peaks of catalpol, rehmannia glycoside D, and chlorogenic acid in the chromatogram of the test solution with the peak areas of the reference standards, the content of catalpol, rehmannia glycoside D, and chlorogenic acid in the test solution can be more easily and accurately detected.

[0025] Preferably, the chromatographic column used in the high-performance liquid chromatography is an Alphasil VC-C18.

[0026] In the above technical solution, by specifically selecting Alphasil VC-C18 as the chromatographic column, it is possible to further and fully separate catalpol, rehmannia glycoside D, and chlorogenic acid in the drug solution, thereby further improving the accuracy of the detection results and making the detection results more stable and reliable.

[0027] Preferably, the column temperature of the chromatographic column is 25–40°C.

[0028] In the above technical solution, by selecting a column temperature of 25-40℃, the separation of catalpol, rehmannia glycoside D, and chlorogenic acid by the Inertsil ODS-3 column can be further promoted, thereby further improving the stability of the detection results.

[0029] Preferably, the mass ratio of Polygonum multiflorum vine, Spatholobus suberectus, Rehmannia glutinosa, Prunus mume, Zanthoxylum bungeanum, Cnidium monnieri, Kochia scoparia, Tribulus terrestris, Dictamnus dasycarpus, and Phellodendron chinense in the medicinal liquid formula is 20:15:30:20:10:20:30:20:20:20.

[0030] In the above technical solution, the ingredients of Polygonum multiflorum vine, Spatholobus suberectus, Rehmannia glutinosa, Prunus mume, Zanthoxylum bungeanum, Cnidium monnieri, Kochia scoparia, Tribulus terrestris, Dictamnus dasycarpus, and Phellodendron chinense are mixed in a mass ratio of 20:15:30:20:10:20:30:20:20, which ensures that the medicinal solution contains catalpol, rehmannia glutinosa D, and chlorogenic acid.

[0031] In summary, this application includes at least one of the following beneficial technical effects:

[0032] This application employs a specific elution procedure: from 0 to 35 minutes, methanol is at 1% and 0.18 wt% phosphoric acid solution is at 99%; from 35 to 45 minutes, methanol increases from 1% to 5% and the 0.18 wt% phosphoric acid solution decreases from 99% to 95%; from 45 to 60 minutes, water increases from 0% to 20%, methanol increases from 5% to 30%, and the 0.18 wt% phosphoric acid solution decreases from 95% to 50%; from 60 to 90 minutes, water decreases from 20% to 0%, methanol increases from 30% to 50%, and the 0.18 wt% phosphoric acid solution is at 50%. This method can simultaneously display the characteristic peaks of catalpol, rehmannia glycoside D, and chlorogenic acid on the chromatogram. It not only eliminates interference from other impurities on the characteristic peaks of catalpol, rehmannia glycoside D, and chlorogenic acid, but also eliminates mutual interference among the characteristic peaks of catalpol, rehmannia glycoside D, and chlorogenic acid. It can better distinguish the characteristic peaks of catalpol, rehmannia glycoside D, and chlorogenic acid, and can accurately detect the content of catalpol, rehmannia glycoside D, and chlorogenic acid in the drug solution based on the peak area. Optimizing the detection of the content of catalpol, rehmannia glycoside D, and chlorogenic acid in the drug solution is conducive to better control of the drug solution quality and further improves the drug solution quality. Attached Figure Description

[0033] Figure 1 The chromatogram of sample 1 at 210 nm is shown.

[0034] Figure 2 The chromatogram of sample 2 at 210 nm is shown.

[0035] Figure 3 The chromatogram of sample 3 at 210 nm is shown.

[0036] Figure 4 The chromatogram of sample 4 at 210 nm is shown.

[0037] Figure 5 The chromatogram of sample 5 at 210 nm is shown.

[0038] Figure 6 The chromatogram of mixed reference solution 1 at 210 nm is shown.

[0039] Figure 7 The chromatogram of the ziziphus alcohol reference solution at 210 nm is shown.

[0040] Figure 8 The chromatogram of the rehmannia glycoside D reference solution at 210 nm is shown.

[0041] Figure 9 The chromatogram of chlorogenic acid reference solution at 210 nm is shown.

[0042] Figure 10 The chromatograms of Method 1 at 320 nm and 236 nm are shown for comparison.

[0043] Figure 11 The chromatograms of Method 2 at 355 nm and 210 nm are shown for comparison.

[0044] Figure 12 The chromatograms of method 3 at 355 nm and 210 nm are shown for comparison.

[0045] Figure 13 The chromatograms of method 4 at 355 nm and 210 nm are shown for comparison.

[0046] Figure 14 The chromatogram at 355 nm is for comparison with method 5.

[0047] Figure 15 The chromatogram at 210 nm is for comparison with method 6. Detailed Implementation

[0048] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0049] instrument:

[0050] Shimadzu analytical balance (AUW 220D), high performance liquid chromatograph (Agilent 1260infinityⅡ), chromatographic column (Alphasil VC-C18).

[0051] Reagents:

[0052] Water (Grade I purified water), methanol (Concord chromatographic reagent), phosphoric acid (analytical grade), ziziphus alcohol (VIP(LY)0998), rehmannia glycoside D (Anzhe Biotechnology A010940-22111), chlorogenic acid (National Institutes for Food and Drug Control 110753-202119).

[0053] Example 1

[0054] A method for simultaneously detecting the contents of catalpol, rehmannia glycoside D, and chlorogenic acid in a drug solution includes the following steps:

[0055] Step 1: Preparation of test solution: In this application, the same drug solution is divided into five parts and each part is prepared into a test solution, which are respectively labeled as sample 1, sample 2, sample 3, sample 4 and sample 5.

[0056] Step 1-1: Preparation of the medicinal liquid: Accurately weigh 20g of Polygonum multiflorum vine, 15g of Spatholobus suberectus, 30g of Rehmannia glutinosa, 20g of Prunus mume, 10g of Zanthoxylum bungeanum, 20g of Cnidium monnieri, 30g of Kochia scoparia, 20g of Tribulus terrestris, 20g of Dictamnus dasycarpus root bark, and 20g of Phellodendron chinense. Soak in 15 times the volume of water for 30 minutes, then decoct at 100℃ for 40 minutes to obtain the medicinal liquid.

[0057] Step 1-2: Add 1 mL of the drug solution to 19 mL of methanol to obtain the test solution.

[0058] Step 2: Preparation of mixed reference solutions: A total of six mixed reference solutions were prepared in this application, which are referred to as mixed reference solution 1, mixed reference solution 2, mixed reference solution 3, mixed reference solution 4, mixed reference solution 5, and mixed reference solution 6, respectively.

[0059] Step 2-1: Accurately weigh 5.41 mg of catalpol, 3.02 mg of rehmannia glycoside D, and 2.56 mg of chlorogenic acid, add them to the diluent and bring the volume to 10 mL in a volumetric flask to prepare the solution.

[0060] The diluent was prepared by mixing methanol and 0.18 wt% phosphoric acid solution in a volume ratio of 1:99.

[0061] Among them, mixed reference solution 1 is the preparation solution.

[0062] Among them, mixed reference solution 2 is prepared by taking 1 mL of preparation solution and making up to 2 mL.

[0063] Among them, mixed reference solution 3 is prepared by taking 1 mL of the preparation solution and making up to 5 mL.

[0064] Among them, mixed reference solution 4 is prepared by taking 1 mL of preparation solution and making up to 10 mL.

[0065] Among them, mixed reference solution 5 is prepared by taking 1 mL of the preparation solution and making up to 20 mL.

[0066] Step 3: Preparation of catalpol reference solution.

[0067] Accurately weigh 5.41 mg of catalpol and add an appropriate amount of diluent to prepare a catalpol reference solution with a concentration of 212.07 ug / ml.

[0068] The diluent was prepared by mixing methanol and 0.18 wt% phosphoric acid solution in a volume ratio of 1:99.

[0069] Step 4: Preparation of Rehmannia glutinosa D reference solution.

[0070] Accurately weigh 3.02 mg of rehmannia glycoside D, add an appropriate amount of diluent, and prepare a catalpol reference solution with a rehmannia glycoside D concentration of 119.78 ug / ml.

[0071] The diluent was prepared by mixing methanol and 0.18 wt% phosphoric acid solution in a volume ratio of 1:99.

[0072] Step 5: Preparation of chlorogenic acid reference solution.

[0073] Accurately weigh 2.56 mg of chlorogenic acid, add an appropriate amount of diluent, and prepare a chlorogenic acid reference solution with a concentration of 98.61 ug / ml.

[0074] The diluent was prepared by mixing methanol and 0.18 wt% phosphoric acid solution in a volume ratio of 1:99.

[0075] Step 6: Construct a chromatogram using high performance liquid chromatography.

[0076] The detection conditions for high performance liquid chromatography are as follows:

[0077] High performance liquid chromatograph: Agilent 1260 Infinity II.

[0078] Chromatographic column: Alphasil VC-C18.

[0079] Column temperature: 25℃.

[0080] Injection volume: 20 μL.

[0081] Flow rate: 0.6 mL / min.

[0082] Detection wavelength: 210nm.

[0083] Mobile phase: water, methanol, 0.18 wt% phosphoric acid solution.

[0084] The gradient elution procedure is as follows:

[0085] From 0 min to 35 min, methanol was 1% and 0.18 wt% phosphoric acid solution was 99%.

[0086] Between 35 and 45 minutes, the methanol concentration increased from 1% to 5%, and the 0.18 wt% phosphoric acid solution decreased from 99% to 95%.

[0087] From 45 to 60 minutes, the water content increased from 0% to 20%, the methanol content increased from 5% to 30%, and the 0.18 wt% phosphoric acid solution decreased from 95% to 50%. From 60 to 90 minutes, the water content decreased from 20% to 0%, the methanol content increased from 30% to 50%, and the 0.18 wt% phosphoric acid solution remained at 50%.

[0088] in, Figure 1 The chromatogram of sample 1 at 210 nm is shown.

[0089] in, Figure 2 The chromatogram of sample 2 at 210 nm is shown.

[0090] in, Figure 3 The chromatogram of sample 3 at 210 nm is shown.

[0091] in, Figure 4 The chromatogram of sample 4 at 210 nm is shown.

[0092] in, Figure 5 The chromatogram of sample 5 at 210 nm is shown.

[0093] in, Figure 6 The chromatogram of mixed reference solution 1 at 210 nm is shown.

[0094] in, Figure 7 The chromatogram of the ziziphus alcohol reference solution at 210 nm is shown.

[0095] in, Figure 8 The chromatogram of the rehmannia glycoside D reference solution at 210 nm is shown.

[0096] in, Figure 9 The chromatogram of chlorogenic acid reference solution at 210 nm is shown.

[0097] Step 7: Calculate the content of effective components in the test solution using the external standard method.

[0098] The peak areas of the three analytes, catalpol, rehmannia glycoside D, and chlorogenic acid, were measured in the mixed reference solution and the test solution, respectively. The contents of the three analytes, catalpol, rehmannia glycoside D, and chlorogenic acid, were calculated according to the following formula:

[0099] Content (ug / mL) = Concentration of test sample * (Peak area or peak height of analyte in chromatogram of test sample solution / Peak area or peak height of analyte in chromatogram of mixed reference solution)

[0100] The results of the measurement of catalpol, rehmannia glycoside D and chlorogenic acid in samples 1-5 are shown in Table 1.

[0101] Table 1:

[0102]

[0103] Example 2

[0104] Repeatability verification:

[0105] According to the content detection results in Table 1, the RSDs of catalpol, rehmannia glycoside D, and chlorogenic acid were 1.33%, 0.88%, and 1.14%, respectively. This indicates that the method has good repeatability and the detection results are stable.

[0106] Example 3

[0107] Authenticity verification:

[0108] Reference Figure 1-9 It can be seen that for samples 1-5, the mixed reference solution, catalpol reference solution, rehmannia glycoside D reference solution and chlorogenic acid reference solution, catalpol, rehmannia glycoside D and chlorogenic acid can all be baseline separated, showing good specificity.

[0109] Example 4

[0110] Linear relationship and range:

[0111] Record the specific peak areas based on the chromatograms of mixed reference solutions 1-6, as detailed in Table 2.

[0112] Table 2:

[0113]

[0114] Standard curves were plotted with peak area as the ordinate (y) and the concentrations of catalpol, rehmannia glycoside D, and chlorogenic acid as the abscissa (x), as shown in Table 3.

[0115] Table 3:

[0116] Element Regression equation <![CDATA[r 2 ]]> <![CDATA[Linear range μg·ml -1 > Zizhu alcohol y = 8.76990x + 1.13895 0.99980 1.06–21.21 Rehmannia glutinosa D y = 10.19075x + 0.05659 0.99986 0.60–11.98 chlorogenic acid y = 65.34050x + 3.15615 0.99979 0.49–9.86

[0117] According to Table 3, catalpol, rehmannia glycoside D, and chlorogenic acid showed good linearity within the range of investigation, with correlation coefficients all greater than 0.999.

[0118] Example 5

[0119] Precision verification:

[0120] Based on the chromatograms of mixed reference solutions 1-6, the RSDs of the peak areas of catalpol, rehmannia glycoside D, and chlorogenic acid were calculated to be 1.95%, 0.20%, and 0.32%, respectively, and the RSDs of the retention times were 1.01%, 0.06%, and 0.05%, respectively. The results indicate that the instrument precision is good.

[0121] Example 6

[0122] Stability verification:

[0123] According to step 1 of Example 1, six test solutions were prepared and stored for 0h, 4h, 8h, 12h, 16h, 20h, and 24h respectively. High performance liquid chromatography was then used for detection. Chromatograms of catalpol, rehmannia glycoside D, and chlorogenic acid were plotted for the test solutions at 0h, 4h, 8h, 12h, 16h, 20h, and 24h respectively, and the contents were calculated. The obtained contents are recorded in Table 4.

[0124] Table 4:

[0125]

[0126] According to Table 4, it can be calculated that catalpol, rehmannia glycoside D, and chlorogenic acid are stable, with RSDs of 0.26%, 1.18%, and 1.84%, respectively, proving that the contents of catalpol, rehmannia glycoside D, and chlorogenic acid are stable within 24 hours.

[0127] Example 7

[0128] Spiked recovery rate

[0129] Two portions of the test solution were prepared according to step 1 of Example 1, and parallel experiments were conducted to determine the spiked recovery rate. Chromatograms were plotted using the high-performance liquid chromatography (HPLC) method described in step 6 of Example 1. Then, 1 mL of each test solution was taken, and 1 mL of each of the reference solutions (cattailol, rehmannia glutinosa D, and chlorogenic acid) was added. Chromatograms were plotted again using the HPLC method described in step 6 of Example 1, and the peak areas were calculated. The spiked recovery rate (average value) was then calculated. The results are shown in Table 5. The calculation method for the spiked recovery rate is as follows:

[0130] Spiked recovery rate (%) = [(Measured value - Sample content) / Amount of reference standard added] * 100%

[0131] Measured value: peak area added;

[0132] Sample content: peak area of ​​sample / 2;

[0133] Add the following amount of reference standard (theoretical value): peak area of ​​reference standard / 2.

[0134] Table 5:

[0135]

[0136] According to Table 5, the recovery rates of catalpol spiked with 94.19%, rehmannia glycoside D spiked with 103.13%, and chlorogenic acid spiked with 110.38% are in line with the recovery rates of 95% to 105% as required by GB / T 27404-2008 "Laboratory Quality Control Standard for Physicochemical Testing of Food".

[0137] The mobile phase and gradient elution program selected for the high-performance liquid chromatography detection conditions in step 6 of Example 1 of this application were compared using the following method:

[0138] Comparison Method 1:

[0139] Mobile phase: water, acetonitrile.

[0140] The gradient elution procedure is as follows:

[0141] From 0 min to 5 min, water accounted for 74% and acetonitrile for 26%;

[0142] Within 5 to 10 minutes, the water content decreased from 74% to 35%, while the acetonitrile content increased from 26% to 65%.

[0143] 10-50 minutes, water 35%, acetonitrile 65%.

[0144] Detection wavelengths: 320nm, 236nm.

[0145] Results Analysis: The high-performance liquid chromatogram is shown below. Figure 10 As shown, no peaks were observed for catalpol, rehmannia glycoside D, and chlorogenic acid, indicating that the three components were not separated.

[0146] Comparison Method 2:

[0147] Since no peak was observed, the detection wavelength was adjusted to 355 nm and 210 nm. The mobile phase and gradient elution program were further adjusted as follows:

[0148] Mobile phase: acetonitrile, 0.2 wt% phosphoric acid.

[0149] The gradient elution procedure is as follows:

[0150] From 0 min to 25 min, acetonitrile was 1%, and phosphoric acid was 99% at 0.2 wt%.

[0151] Between 25 and 35 minutes, acetonitrile increased from 1% to 18%, while 0.2 wt% phosphoric acid decreased from 99% to 82%.

[0152] Between 35 and 45 minutes, acetonitrile increased from 18% to 50%, while 0.2 wt% phosphoric acid decreased from 82% to 50%.

[0153] 45 min–55 min, acetonitrile 50%, 0.2 wt% phosphoric acid 50%.

[0154] Detection wavelengths: 355nm, 210nm.

[0155] Results Analysis: The high-performance liquid chromatogram is shown below. Figure 11 As shown, peaks of catalpol, rehmannia glycoside D, and chlorogenic acid were observed at 210 nm, but the separation of each peak was not obvious.

[0156] Comparison Method 3:

[0157] The results of method 2 were unsatisfactory, so the mobile phase was further adjusted by replacing acetonitrile with methanol, as shown below:

[0158] Mobile phase: methanol, 0.2 wt% phosphoric acid.

[0159] The gradient elution procedure is as follows:

[0160] From 0 min to 35 min, methanol was 1% and phosphoric acid was 99% (0.2 wt%).

[0161] Between 35 and 45 minutes, methanol increased from 1% to 30%, while 0.2 wt% phosphoric acid decreased from 99% to 70%.

[0162] Between 45 and 65 minutes, methanol increased from 30% to 50%, while 0.2 wt% phosphoric acid decreased from 70% to 50%.

[0163] The reaction time was 65-80 minutes, with 50% methanol and 50% 0.2 wt% phosphoric acid.

[0164] Detection wavelengths: 355nm, 210nm.

[0165] Results Analysis: The high-performance liquid chromatogram is shown below. Figure 12 As shown, the peak separation effect is not obvious at 355nm and 210nm.

[0166] Comparison Method 4:

[0167] The results of method 3 were unsatisfactory, so the gradient elution procedure was further adjusted as follows:

[0168] Mobile phase: methanol, 0.2 wt% phosphoric acid.

[0169] The gradient elution procedure is as follows:

[0170] From 0 min to 25 min, methanol was 1% and phosphoric acid was 99% (0.2 wt%).

[0171] Between 25 and 40 minutes, methanol increased from 1% to 5%, while 0.2 wt% phosphoric acid decreased from 99% to 95%.

[0172] Between 40 and 55 minutes, methanol increased from 5% to 30%, while 0.2 wt% phosphoric acid decreased from 95% to 70%.

[0173] Between 55 and 65 minutes, methanol increased from 30% to 50%, while 0.2 wt% phosphoric acid decreased from 70% to 50%.

[0174] The reaction time was 65-80 minutes, with 50% methanol and 50% 0.2 wt% phosphoric acid.

[0175] Detection wavelengths: 355nm, 210nm.

[0176] Results Analysis: The high-performance liquid chromatogram is shown below. Figure 13 As shown, the peak separation effect is not obvious at 355nm and 210nm.

[0177] Comparison Method 5:

[0178] The results of method 4 were unsatisfactory, so the gradient elution procedure was further adjusted as follows:

[0179] Mobile phase: methanol, 0.4 wt% phosphoric acid.

[0180] The gradient elution procedure is as follows:

[0181] From 0 min to 20 min, methanol was 18% and 0.4 wt% phosphoric acid was 82%.

[0182] Between 20 and 30 minutes, methanol increased from 18% to 30%, while 0.4 wt% phosphoric acid decreased from 82% to 70%.

[0183] Within 30 to 40 minutes, the methanol content increased from 30% to 50%, while the 0.4 wt% phosphoric acid content decreased from 70% to 50%.

[0184] 40 min to 60 min, methanol 50%, 0.4 wt% phosphoric acid 50%.

[0185] Detection wavelength: 355nm.

[0186] Results Analysis: The high-performance liquid chromatogram is shown below. Figure 14 As shown, the peak separation effect is not obvious at 355nm.

[0187] Comparison Method 6:

[0188] The results of method 5 were still unsatisfactory. Further adjustments were made to the gradient elution procedure and detection wavelength, as shown below:

[0189] Mobile phase: methanol, 0.2 wt% phosphoric acid solution.

[0190] The gradient elution procedure is as follows:

[0191] From 0 min to 35 min, methanol was 1% and 0.2 wt% phosphoric acid solution was 99%;

[0192] Between 35 and 45 minutes, the methanol concentration increased from 1% to 5%, and the 0.2 wt% phosphoric acid solution decreased from 99% to 95%.

[0193] Over 45 to 60 minutes, the methanol concentration increased from 5% to 30%, while the concentration of the 0.2 wt% phosphoric acid solution decreased from 95% to 70%.

[0194] Over 60 to 90 minutes, the methanol concentration increased from 30% to 50%, while the 0.2 wt% phosphoric acid solution decreased from 70% to 50%.

[0195] Detection wavelength: 210nm.

[0196] Results Analysis: The high-performance liquid chromatogram is shown below. Figure 15 As shown, the peaks are well separated, but the peak shapes are not obvious, which may lead to excessive errors in the detection results.

[0197] Analysis of the chromatograms from methods 1 to 6 shows that the selection of the mobile phase and gradient elution program for high-performance liquid chromatography (HPLC) detection conditions is somewhat accidental. Any deviation in these conditions will affect the final chromatogram results. The mobile phase and gradient elution program selected for HPLC detection conditions in Example 1 of this application unexpectedly achieved the desired results: peaks of citronellol, rehmannia glycoside D, and chlorogenic acid were clearly visible, with good separation of each peak and distinct peak shapes.

[0198] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for simultaneously detecting the contents of catalpol, rehmannia glycoside D, and chlorogenic acid in a pharmaceutical solution, characterized in that, The medicinal solution is prepared by decocting ten medicinal materials, including Polygonum multiflorum vine, Spatholobus suberectus, Rehmannia glutinosa, Prunus mume, Zanthoxylum bungeanum, Cnidium monnieri, Kochia scoparia, Tribulus terrestris, Dictamnus dasycarpus, and Phellodendron chinense. The detection method includes the following steps: preparation of the test solution, preparation of the mixed reference solution, and detection by high performance liquid chromatography. The mobile phase in the high-performance liquid chromatography detection conditions includes water, methanol, and 0.18 wt% phosphoric acid solution; The high-performance liquid chromatography method employs a gradient elution procedure; The gradient elution procedure is as follows: 0 min ~ 35 min, methanol 1%, 0.18 wt% phosphoric acid solution 99%; Between 35 and 45 minutes, the methanol content increased from 1% to 5%, while the 0.18 wt% phosphoric acid solution decreased from 99% to 95%. Over 45-60 minutes, the concentration of water increased from 0% to 20%, methanol increased from 5% to 30%, and the concentration of 0.18 wt% phosphoric acid solution decreased from 95% to 50%. Over 60-90 minutes, the water content decreased from 20% to 0%, the methanol content increased from 30% to 50%, and the 0.18 wt% phosphoric acid solution reached 50%. The detection wavelength in the high-performance liquid chromatography detection conditions is 210 nm; The test solution is a drug solution diluted with methanol, and the volume ratio of the drug solution to methanol is (1 mL~2 mL): (18 mL~19 mL). The chromatographic column used in the high-performance liquid chromatography method is an Alphasil VC-C18.

2. The method for simultaneously detecting the contents of catalpol, rehmannia glycoside D, and chlorogenic acid in a drug solution according to claim 1, characterized in that, The flow rate in the high-performance liquid chromatography detection conditions is 0.6 mL / min.

3. The method for simultaneously detecting the contents of catalpol, rehmannia glycoside D, and chlorogenic acid in a drug solution according to claim 1, characterized in that, The injection volume in the high-performance liquid chromatography detection conditions is 15~20 μL.

4. The method for simultaneously detecting the contents of catalpol, rehmannia glycoside D, and chlorogenic acid in a drug solution according to claim 1, characterized in that, The preparation method of the mixed reference solution for high performance liquid chromatography is as follows: accurately weigh appropriate amounts of catalpol reference standard, rehmannia glutinosa D reference standard and chlorogenic acid reference standard and mix them to obtain a mixture. Add an appropriate amount of diluent to the mixture to prepare the mixed reference solution. The diluent was prepared by mixing methanol and 0.18 wt% phosphoric acid solution in a volume ratio of 1:

99.

5. The method for simultaneously detecting the contents of catalpol, rehmannia glycoside D, and chlorogenic acid in a drug solution according to claim 1, characterized in that, The column temperature of the chromatographic column is 25~40℃.

6. The method for simultaneously detecting the contents of catalpol, rehmannia glycoside D, and chlorogenic acid in a drug solution according to claim 1, characterized in that, The mass ratio of Polygonum multiflorum vine, Spatholobus suberectus, Rehmannia glutinosa, Prunus mume, Zanthoxylum bungeanum, Cnidium monnieri, Kochia scoparia, Tribulus terrestris, Dictamnus dasycarpus, and Phellodendron chinense in the medicinal liquid formula is 20:15:30:20:10:20:30:20:20:20.

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