A method for simultaneously determining the contents of 12 index components in Renshen Jianpi Pills using HPLC wavelength switching technology

Abstract

The invention discloses a method for simultaneously determining the contents of 12 index components in ginseng Jianpi pills by using HPLC wavelength switching technology. Specifically, HPLC wavelength switching technology is used to determine cryptochlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-dicerinoyl sucrose, hesperidin, nobiletin, tangeretin, ligustilide, and atractylodes lactone I in ginseng Jianpi pills. The HPLC detection wavelength is: 0-14min, 325nm; 14-20min, 260nm; 20-21.5min, 325nm; 21.5-22.5min, 282nm; 22.5-60min, 325nm; 60-70min, 277nm. The method of the invention can simultaneously perform qualitative and quantitative detection on 12 components in ginseng Jianpi pills, and the detection sensitivity is improved by using HPLC wavelength switching technology, and the measurement result is more accurate, which reflects the quality of ginseng Jianpi pills as a whole.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and specifically relates to a method for simultaneously determining the contents of 12 index components in ginseng invigorating spleen pills using an HPLC wavelength switching technology. Background Art

[0002] Ginseng Jianpi Pills originated from Wang Kentang's "Zhengzhi Zhunsheng·Lianfang" in the Ming Dynasty. It is now included in the national drug standards of the 2020 edition of the "Chinese Pharmacopoeia". It is composed of 11 herbs: ginseng, Atractylodes macrocephala (fried with bran), Poria cocos, Chinese yam, dried tangerine peel, costus root, Amomum villosum, roasted Astragalus, Angelica sinensis, Ziziphus jujuba seeds (fried), and Polygala tenuifolia (processed). All of them are raw powders of medicinal materials. It has the effects of tonifying the spleen and replenishing qi, harmonizing the stomach and stopping diarrhea. It is mainly used clinically for indigestion, abdominal distension and noise, nausea and vomiting, abdominal pain and loose stools, loss of appetite, and physical weakness and fatigue caused by weak spleen and stomach. The current quality standard of ginseng Jianpi Pills has three specifications: large honey pills, small honey pills, and water honey pills, but generally only includes the content determination of one ingredient, hesperidin, which cannot reflect the quality status of the product as a whole. In addition, there is currently little research on the material basis of ginseng Jianpi Pills, and there are many manufacturers. There is no literature report on the difference in product quality between different manufacturers and different production batches.

[0003] Chinese patent CN 115541768A discloses a detection method for simultaneously identifying eight raw materials in ginseng jianpi pills, using UPLC-Q-Orbitrap high performance liquid chromatography-mass spectrometry to simultaneously identify eight raw materials in ginseng jianpi pills, including ginseng, tangerine peel, costusroot, astragalus, angelica, polygala, atractylodes and spiny jujube seeds. This method uses characteristic peak retention time and accurate mass number as judgment criteria, but the characteristic peak retention times of different raw materials are very close, such as the ginseng characteristic peak at 16.18min, the spiny jujube seed characteristic peak at 16.80min, the polygala characteristic peak at 16.99min, the astragalus characteristic peak at 17.17min, and the spiny jujube seed characteristic peak at 17.51min. The peaks here are relatively dense, and deviations are likely to occur, and quantitative detection of a certain raw material component is not achieved.

[0004] Therefore, it is of great significance to develop a method for evaluating the quality of ginseng Jianpi pills with simple testing method and high accuracy. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for simultaneously determining the contents of 12 index components in ginseng jianpi pills by HPLC wavelength switching technology. The test method is simple and has high accuracy, and can reflect the quality of ginseng jianpi pills as a whole.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for simultaneously determining the contents of 12 index components in ginseng jianpi pills by using HPLC wavelength switching technology. The HPLC wavelength switching technology is used to determine the contents of cryptochlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-diesinapoylsucrose, hesperidin, nobiletin, tangeretin, ligustilide and atractylodes lactone I in the ginseng jianpi pills. The HPLC detection wavelengths are: 0-14 min, 325 nm; 14-20 min, 260 nm; 20-21.5 min, 325 nm; 21.5-22.5 min, 282 nm; 22.5-60 min, 325 nm; 60-70 min, 277 nm.

[0008] Furthermore, the method for simultaneously determining the contents of 12 index components in Ginseng Jianpi Pills by using the HPLC wavelength switching technology specifically comprises the following steps:

[0009] (1) Preparation of mixed reference solution: Weigh out chlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-diesinapoylsucrose, hesperidin, nobiletin, tangeretin, ligustilide, and atractylodes lactone I reference substances respectively, and add methanol to prepare a solution containing 17.75 μg chlorogenic acid, 20.02 μg vanillic acid, 19.9 μg spinosin, and 10.5 μg atractylodes lactone per 1 mL. A mixed reference solution of 6 μg, 30.81 μg of calycosin glucoside, 21.07 μg of ferulic acid, 84.14 μg of rutin, 44.24 μg of 3,6'-diesinapoylsucrose, 638.38 μg of hesperidin, 17.63 μg of nobiletin, 17.17 μg of tangeretin, 162.48 μg of ligustilide, and 17.28 μg of atractylodes lactone;

[0010] (2) Preparation of test solution: Take 2-3 g of Ginseng Jianpi Pills, add methanol, heat under reflux in a water bath for 0.5-2 h, take out, cool, weigh, make up the lost weight with methanol, shake well, filter, and obtain the test solution;

[0011] (3) Drawing of standard curve: The mixed reference substance solution was diluted into different concentration gradients, injected into HPLC, and detected by wavelength switching method. The standard curve was drawn with mass concentration as the horizontal axis (X) and peak area integral value as the vertical axis (Y);

[0012] (4) Test sample detection: The test sample solution is injected into HPLC and detected by wavelength switching method. The position and peak area of ​​each chromatographic peak are determined and substituted into the standard curve to calculate the content of each component in the test sample.

[0013] Furthermore, the ginseng spleen-strengthening pills described in step (2) are one of water-honeyed pills, small honeyed pills and large honeyed pills.

[0014] Furthermore, the HPLC chromatographic conditions are as follows: the chromatographic column is Kromasil 100-5-C 18 , 250mm×4.6mm5μm, mobile phase is acetonitrile-0.1% formic acid aqueous solution, gradient elution, 0~5min, 5% acetonitrile; 5~60min, 5%→65% acetonitrile; 60~63min, 65% acetonitrile; 63~66min, 65%→90% acetonitrile; 66~70min, 90% acetonitrile; flow rate is 1.0mL·min -1 ; Column temperature was 35°C; Injection volume was 10 μL.

[0015] Furthermore, in step (2), the large honey pills or small honey pills are added with diatomaceous earth, fully ground and then cut into pieces, and then methanol is added, and the mass ratio of the large honey pills or small honey pills to the diatomaceous earth is 2 to 3:1.5.

[0016] Beneficial Effects

[0017] The method of the present invention can simultaneously perform qualitative and quantitative detection on the components of cryptochlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-diesinapoylsucrose, hesperidin, nobiletin, tangeretin, ligustilide and atractylodes lactone I12 in the ginseng invigorating spleen pills, and utilizes HPLC wavelength switching technology to improve the detection sensitivity, so that the measurement result is more accurate and can reflect the quality of the ginseng invigorating spleen pills as a whole. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the HPLC chromatogram of the mixed reference solution;

[0019] Figure 2 This is the HPLC chromatogram of the Ginseng Jianpi Pill sample. DETAILED DESCRIPTION

[0020] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only to further illustrate the features and advantages of the present invention, rather than to limit the present invention.

[0021] Instruments, reagents and samples in the embodiments of the present invention:

[0022] Waters 2695 high performance liquid chromatograph (UV detector, Waters, USA); XSE205 1 / 100000 electronic balance (METTLER TOLEDO, Germany);

[0023] Reference substances: vanillic acid (110776-200402, 100.0%), spinosin (111869-202005, 94.8%), calycosin glucoside (111920-201907, 96.8%), ferulic acid (110773-201915, 99.4%), 3,6'-bisinacoylsucrose (111848-202006, 96.5%) , hesperidin (110721-201818, 96.2%), nobiletin (112055-202001, 99.6%), tangeretin (112054-202102, 100.0%), ligustilide (111737-201910, 100.0%), and atractylodes lactone I (111975-201501, 99.9%) were purchased from the China Food and Drug Administration. Naringin (batch number MUST-22040211, 98.76%) and cryptochlorogenic acid (batch number MUST-16022403, 99.07%) were purchased from Chengdu Pusi Biotechnology Co., Ltd.

[0024] The samples are national drug random inspection samples in 2022, totaling 137 batches. For specific information, see Table 9.

[0025] Acetonitrile (Honeywell B&J) and formic acid (Shanghai Aladdin Biotechnology Co., Ltd.) were all chromatographically pure; the other reagents were all analytically pure (Tianjin Komiou Chemical Reagent Co., Ltd.); water was prepared by Milli-Q Advantage A10 ultrapure water instrument (Millipore, USA).

[0026] Example 1

[0027] (1) Preparation of mixed reference solution: Weigh chlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-dicerinoylsucrose,

[0028] Hesperidin, nobiletin, tangeretin, ligustilide, and atractylodes lactone I reference substances were prepared by adding methanol to prepare a solution containing 17.75 μg of cryptochlorogenic acid, 20.02 μg of vanillic acid, and 20.02 μg of spinosin per mL.

[0029] 19.96μg, calycosin glucoside 30.81μg, ferulic acid 21.07μg, naringin 84.14μg, 3,6'-dicorinoylsucrose 44.24μg, hesperidin

[0030] A mixed reference solution of 638.38μg, nobiletin 17.63μg, tangeretin 17.17μg, ligustilide 162.48μg, and atractylodes lactone 17.28μg;

[0031] (2) Preparation of test solution: Take an appropriate amount of honey-coated pills, grind them, and accurately weigh 2 g (or take an appropriate amount of small honey-coated pills or large honey-coated pills, cut them into pieces, accurately weigh 3 g, add 1.5 g of diatomaceous earth, grind them into thin slices, and cut them into pieces), place them in a round-bottom flask, accurately add 25 mL of methanol, seal it tightly, weigh it, heat it under reflux in a water bath for 1 hour, take it out, let it cool, weigh it again, make up the lost weight with methanol, shake it well, and filter it to obtain the test solution;

[0032] (3) Drawing of standard curve: Accurately measure 20 mL of the mixed reference solution prepared in step (1),

[0033] 10mL, 5mL, 2mL, and 1mL were placed in 20mL volumetric flasks, methanol was added to the mark, and shaken well. 10μL of each series of reference solution was accurately pipetted and injected into the high performance liquid chromatograph for determination.

[0034] The concentration is the horizontal axis (X), the peak area integral value is the vertical axis (Y), and the standard curve is drawn. The HPLC chromatogram of the mixed reference solution is as follows: Figure 1 As shown;

[0035] The chromatographic column is Kromasil 100-5-C 18 (250 mm × 4.6 mm, 5 μm); acetonitrile-0.1%

[0036] Formic acid aqueous solution was used as the mobile phase, and gradient elution (0-5 min, 5% acetonitrile; 5-60 min, 5% → 65%) was used.

[0037] Acetonitrile; 60-63 min, 65% acetonitrile; 63-66 min, 65%→90% acetonitrile; 66-70 min, 90% acetonitrile); flow rate: 1.0 mL min -1 ; Column temperature was 35°C; Wavelength switching detection (0-14min,

[0038] 325nm; 14~20min, 260nm; 20~21.5min, 325nm; 21.5~22.5min, 282nm;

[0039] 22.5~60min, 325nm; 60~70min, 277nm); the regression equations, correlation coefficients (r) and mass concentration linear ranges of the 12 index components are shown in Table 1.

[0040] Table 1 Linear relationship table of 12 index components

[0041] Element <![CDATA[Linear range of mass concentration ( μg / ml)]]> Regression equation Correlation coefficient r <![CDATA[ Cryptochlorogenic acid ]]> 0.89～17.75 y=24715.38x-1234.44 0.9997 Vanillic acid 1.00～20.02 y=40344.38x-9293.48 1.0000 <![CDATA[Si Pinus > 1.00～19.96 y=17685.65x-4017.61 0.9999 Isoflavone Glucoside 1.54～30.81 y=33153.30x+2374.01 1.0000 Ferulic acid 1.05～21.07 y=58843.94x-12207.33 1.0000 <![CDATA[Citrus grandis 'Tomentosa' peel Glycoside > 4.21～84.14 y=18734.80x-14875.29 1.0000 3,6'-Dierucyl Sucrose 2.21～44.24 y=25744.38x-10738.31 0.9999 <![CDATA[Orange peel Glycoside > 31.92～638.38 y=3307.10x-20258.49 1.0000 Nobiletin 0.88～17.63 y=39227.31x-8373.94 0.9999 Tangeretin 0.86～17.17 y=56682.12x-6972.39 0.9999 <![CDATA[ Gao Ben lactone]]> 8.12～162.48 y=11360.65x-17617.90 1.0000 Atractylodes lactone I 0.86～17.28 y=44520.28x-4131.79 0.9998

[0042] (4) Test sample detection: inject the test sample solution prepared in step (2) into a high performance liquid chromatograph, determine the chromatographic peak position and peak area of ​​each component, substitute into the standard curve, and calculate the content of each component in the test sample; the detection conditions are the same as those in step (3). The HPLC chromatogram of the Renshen Jianpi Pill sample is as follows: Figure 2 shown.

[0043] Example 2

[0044] Precision test

[0045] 10 μL of the mixed reference solution of step (1) of Example 1 was accurately drawn and injected 6 times in succession. The RSD of the peak area integral values ​​of cryptochlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-dicerinoylsucrose, hesperidin, nobiletin, tangeretin, ligustilide, and atractylodes lactone I were all <2%, indicating that the instrument had good precision. The results are shown in Table 2 below.

[0046] Table 2 Precision test results

[0047] Element 1 2 3 4 5 6 average value <![CDATA[ RSD (%)]]> <![CDATA[ Cryptochlorogenic acid ]]> 225752 225550 225008 228943 222207 224346 225301 0.97 Vanillic acid 403519 401761 402041 391924 398697 396291 399039 1.09 <![CDATA[Si Pinus > 176518 176953 175668 176680 173940 173374 175522 0.86 Isoflavone Glucoside 516058 511987 513364 517007 513234 515566 514536 0.38 Ferulic acid 620638 620655 612506 618317 608011 607743 614645 0.98 <![CDATA[Citrus grandis 'Tomentosa' peel Glycoside > 788216 783589 776565 781340 775503 775800 780169 0.66 3,6'-Dierucyl Sucrose 554855 564282 557505 560588 551862 563497 558765 0.88 <![CDATA[Orange peel Glycoside > 1044477 1051112 1033711 1044110 1038914 1032594 1040820 0.68 Nobiletin 346444 344632 341823 330901 332932 339357 339348 1.85 Tangeretin 389462 383339 376591 376645 379790 380355 381030 1.27 <![CDATA[ Gao Ben lactone]]> 923542 927327 902839 917012 913154 906824 915116 1.03 Atractylodes lactone I 494548 489312 477861 480740 473722 474180 481727 1.76

[0048] Example 3

[0049] Stability test

[0050] The same test solution (prepared according to the sample preparation method of Example 1) was accurately drawn, and 10 μL was injected at 0, 6, 12, 18, and 24 hours, respectively, to determine the peak area. The results showed that the RSD of the peak area integral values ​​of cryptochlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, rutin, 3,6'-bisinacoyl sucrose, hesperidin, nobiletin, tangeretin, ligustilide, and atractylodes lactone I were all <2%. This shows that the 12 index components to be tested in the test solution are basically stable within 24 hours at room temperature. The results are shown in Tables 3 and 4 below.

[0051] Table 3 Stability test results (large honey pills, Shandong Hongjitang Pharmaceutical Group Co., Ltd., batch number 2203002)

[0052] Element 0h 6h 12h 18h 24h average value <![CDATA[ RSD (%)]]> <![CDATA[ Cryptochlorophenol ]]> 57346 57199 57425 57602 57101 57335 0.34 Vanillic acid 81718 81093 81425 81169 81672 81415 0.35 <![CDATA[S Pinus > 85543 84842 85187 86448 86451 85694 0.86 Isoflavone Glucoside 102922 100649 102079 100355 102347 89002 1.25 Ferulic acid 231314 236655 231602 230305 228298 231635 1.33 <![CDATA[Citrus grandis 'Tomentosa' peel Glycoside > 719858 712576 722382 724596 724612 720805 0.69 3,6'-Dierucyl Sucrose 306110 309961 311185 312142 312149 310309 0.81 <![CDATA[Orange peel Glycoside > 814306 808180 811387 813861 813879 812323 0.32 Nobiletin 57269 57125 57350 57525 58028 57459 0.61 Tangeretin 34669 34189 34325 34432 34435 34410 0.51 <![CDATA[ Gao Ben lactone]]> 668521 670127 667802 669827 669835 669222 0.15 Atractylodes lactone I 166248 168905 167578 168090 170099 168184 0.86

[0053] Table 4 Stability test results (water-honeyed pills, Shandong Hongjitang Pharmaceutical Group Co., Ltd., batch number 2203007)

[0054] Element 0h 6h 12h 18h 24h average value <![CDATA[ RSD (%)]]> <![CDATA[ Cryptochlorogenic acid ]]> 82511 81215 81777 82072 81384 81792 0.64 Vanillic acid 66092 64496 65460 66165 65159 65474 1.06 <![CDATA[Si Pinus > 74985 73516 73784 74646 73527 74092 0.92 Isoflavone Glucoside 89793 89762 89888 89125 89878 89002 0.36 Ferulic acid 185133 177967 178222 179955 183184 180892 1.74 <![CDATA[Citrus grandis 'Tomentosa' peel Glycoside > 860974 847346 855701 844542 854952 852703 0.78 3,6'-Dierucyl succinate 447173 443767 444736 441518 441738 443786 0.52 <![CDATA[Orange peel Glycoside > 918875 915517 913301 914648 911387 914746 0.30 Nobiletin 59812 58542 59604 59701 59284 59389 0.86 Tangeretin 37366 36586 36964 37680 36653 37050 1.26 <![CDATA[ Gao Ben lactone]]> 727074 724030 720617 721233 719049 722401 0.44 Atractylodes lactone I 161647 162392 161185 162306 160379 161582 0.52

[0055] Example 4

[0056] Repeatability test

[0057] (1) Take an appropriate amount of the test sample (Shandong Hongjitang Pharmaceutical Group Co., Ltd., large honey pills, batch number 2203002), and prepare 6 test sample solutions in parallel according to the preparation method of the test sample solution in step (2) of Example 1. Inject 10 μL of each solution according to the chromatographic conditions of Example 1, measure the peak area, and calculate the contents of the 12 index components according to the linear equation obtained in Example 1. Results The average contents of cryptochlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-diesinapoylsucrose, hesperidin, nobiletin, tangeretin, ligustilide and atractylodes lactone I in the samples were 0.020, 0.019, 0.042, 0.025, 0.035, 0.326, 0.104, 2.092, 0.014, 0.006, 0.503 and 0.032 mg / g, respectively, and the RSDs were all <2%, indicating good repeatability. The results are shown in Table 5.

[0058] Table 5 Repeatability test results (large honey pills)

[0059] Element 1 2 3 4 5 6 Average value (mg / g) <![CDATA[ RSD (%)]]> <![CDATA[ Cryptochlorophenol ]]> 0.0199 0.0197 0.0197 0.0198 0.0197 0.0196 0.0197 0.52 Vanillic acid 0.0187 0.0187 0.0187 0.0188 0.0185 0.0187 0.0187 0.53 <![CDATA[Si Pinus > 0.0420 0.0421 0.0420 0.0421 0.0422 0.0425 0.0422 0.44 Isoflavone Glucoside 0.0250 0.0252 0.0248 0.0251 0.0244 0.0250 0.0249 1.15 Ferulic acid 0.0347 0.0344 0.0353 0.0346 0.0340 0.0339 0.0345 1.48 <![CDATA[Citrus grandis 'Tomentosa' peel Glycoside > 0.3260 0.3257 0.3243 0.3283 0.3260 0.3278 0.3264 0.45 36'-Dierucyl Sucrose 0.1031 0.1022 0.1040 0.1043 0.1036 0.1042 0.1036 0.77 <![CDATA[Orange peel Glycoside > 2.0898 2.0956 2.0923 2.0979 2.0830 2.0946 2.0922 0.25 Nobiletin 0.0140 0.0139 0.0139 0.0140 0.0139 0.0141 0.0140 0.58 Tangeretin 0.0060 0.0061 0.0061 0.0061 0.0060 0.0061 0.0061 0.85 <![CDATA[ Gao Ben lactone]]> 0.5062 0.5015 0.5056 0.5033 0.4997 0.5025 0.5031 0.49 Atractylodes lactone 1 0.0321 0.0318 0.0325 0.0322 0.0319 0.0325 0.0322 0.91

[0060] (2) Take an appropriate amount of the test sample (Shandong Hongjitang Pharmaceutical Group Co., Ltd., water-honeyed pills, batch number 2203007), and prepare 6 test sample solutions in parallel according to the preparation method of the test sample solution in step (2) of Example 1. Inject 10 μL of each solution according to the chromatographic conditions of Example 1, measure the peak area, and calculate the contents of the 12 index components according to the linear equation obtained in Example 1. Results The average contents of cryptochlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-diesinapoylsucrose, hesperidin, nobiletin, tangeretin, ligustilide and atractylodes lactone I in the samples were 0.042, 0.023, 0.054, 0.034, 0.041, 0.577, 0.220, 3.520, 0.022, 0.010, 0.811 and 0.046 mg / g, respectively, and the RSDs were all <2%, indicating good repeatability. The results are shown in Table 6.

[0061] Table 6 Repeatability test results (water-honey pills)

[0062] Element 1 2 3 4 5 6 Average value (mg / g) <![CDATA[ RSD (%)]]> <![CDATA[ Cryptochlorogenic acid ]]> 0.0419 0.0417 0.0417 0.0416 0.0419 0.0418 0.0418 0.29 Vanillic acid 0.0231 0.0233 0.0230 0.0225 0.0229 0.0232 0.0230 1.23 <![CDATA[Si Pinosum > 0.0552 0.0531 0.0542 0.0542 0.0548 0.0550 0.0544 1.41 Isoflavone Glucoside 0.0334 0.0334 0.0337 0.0334 0.0332 0.0336 0.0335 0.53 Ferulic acid 0.0413 0.0414 0.0404 0.0399 0.0409 0.0404 0.0407 1.44 <![CDATA[Citrus grandis 'Tomentosa' peel Glycoside > 0.5781 0.5829 0.5779 0.5777 0.5663 0.5762 0.5765 0.95 36'-Dierucyl Sucrose 0.2199 0.2194 0.2192 0.2234 0.2197 0.2204 0.2203 0.71 <![CDATA[Orange peel Glycoside > 3.5116 3.5096 3.5137 3.5550 3.5090 3.5207 3.5199 0.50 Nobiletin 0.0215 0.0213 0.0216 0.0219 0.0215 0.0209 0.0215 1.55 Tangeretin 0.0097 0.0096 0.0095 0.0097 0.0094 0.0097 0.0096 1.32 <![CDATA[ Gao Ben lactone]]> 0.8106 0.8144 0.8097 0.8187 0.8030 0.8107 0.8112 0.64 Atractylodes lactone I 0.0460 0.0458 0.0464 0.0469 0.0465 0.0468 0.0464 0.93

[0063] Example 5

[0064] Sample recovery test

[0065] (1) Take an appropriate amount of the test sample (Shandong Hongjitang Pharmaceutical Group Co., Ltd., large honey pills, batch number 2203002) with a known content, cut it into pieces, take 1.5 g, accurately weigh it, add 0.75 g of diatomaceous earth, grind it into thin slices, cut it into pieces, put it in a round-bottom flask, and accurately add the mixed reference solution (cryptochlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-dicerinoylsucrose, hesperidin, nobiletin, tangeretin, ligusticum wallichii) The concentrations of lactone and Atractylodes lactone I were 3.057, 2.817, 6.258, 3.722, 5.159, 50.077, 15.223, 327.083, 2.203, 1.001, 81.240, and 4.320 μg / mL, respectively) 10 mL, then accurately add 15 mL of methanol, weigh the weight, heat under reflux for 1 hour, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0066] Six test solutions were prepared in parallel, and 10 μL was injected according to the chromatographic conditions of Example 1, and the peak area was determined. The recoveries of the 12 index components were calculated according to the linear equation in Table 1. The results showed that the average recoveries of chlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-bisinacoylsucrose, hesperidin, nobiletin, tangeretin, ligustilide, and atractylodes lactone I (n=6) were 98.34%, 102.41%, 103.83%, 97.19%, 103.16%, 97.15%, 98.62%, 96.40%, 99.54%, 93.66%, 91.28%, and 95.16%, respectively; RSDs were all <2%, indicating that the recoveries were good. The results are shown in Table 7.

[0067] Table 7 Sample recovery test results (large honey pills, unit: %)

[0068] Element 1 2 3 4 5 6 average value RSD <![CDATA[ Cryptochlorophenol ]]> 98.67 99.19 97.55 98.27 97.81 98.53 98.34 0.61 Vanillic acid 102.61 102.63 102.41 101.79 102.37 102.63 102.41 0.32 <![CDATA[Si Pinosum > 102.98 104.89 102.33 104.37 103.99 104.41 103.83 0.94 Isoflavone Glucoside 97.82 98.74 95.62 97.01 97.52 96.45 97.19 1.12 Ferulic acid 103.87 102.94 103.77 103.19 102.69 102.51 103.16 0.54 <![CDATA[Citrus grandis 'Tomentosa' peel Glycoside > 97.24 98.26 96.51 97.06 96.54 97.30 97.15 0.66 3,6'-Dierucyl Sucrose 97.49 99.37 97.16 99.76 98.79 99.17 98.62 1.07 <![CDATA[Orange peel Glycoside > 97.82 97.26 95.66 95.79 95.64 96.25 96.40 0.96 Nobiletin 101.44 101.28 99.38 98.52 98.32 98.31 99.54 1.47 Tangeretin 95.41 92.94 92.79 92.42 93.76 94.66 93.66 1.25 <![CDATA[ Gao Ben lactone]]> 91.42 91.01 91.06 91.41 92.37 90.42 91.28 0.71 Atractylodes lactone I 97.23 95.85 94.81 94.42 95.42 93.24 95.16 1.42

[0069] (2) Take an appropriate amount of the test sample (Shandong Hongjitang Pharmaceutical Group Co., Ltd., Shuimi Pills, batch number 2203007) with a known content, grind it, take about 1 g, weigh it accurately, and add it accurately to the mixed reference solution (the concentrations of cryptochlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-diesinapoylsucrose, hesperidin, nobiletin, tangeretin, ligustilide, and atractylodes lactone I are respectively 4.272, 2.338, 5.530, 3.081, 4.138, 56.096, 22.118, 354.660, 2.203, 1.001, 81.240, 4.320 μg / mL) 10mL, then accurately add 15mL of methanol, weigh the weight, heat under reflux for 1 hour, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

[0070] Six test solutions were prepared in parallel, and 10 μL was injected according to the chromatographic conditions of Example 1, and the peak area was determined. The recovery rates of the 12 index components were calculated using the linear equation in Table 1. Results The average recovery rates (n=6) of cryptochlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-bisinacoylsucrose, hesperidin, nobiletin, tangeretin, ligustilide, and atractylodes lactone I were 97.83%, 103.80%, 104.91%, 96.76%, 104.89%, 96.74%, 98.35%, 95.90%, 99.61%, 93.25%, 90.55%, and 94.74%, respectively; RSDs were all <2%, indicating that the recovery rates were good. The results are shown in Table 8.

[0071] Table 8 Sample recovery test results (water-honeyed pills, unit: %)

[0072] Element 1 2 3 4 5 6 average value RSD <![CDATA[ Cryptochlorogenic acid ]]> 97.58 99.19 97.1 97.93 97.06 98.11 97.83 0.81 Vanillic acid 103.57 104.11 102.51 103.48 104.42 104.71 103.8 0.76 <![CDATA[Si Pinosum > 102.95 106.47 102.43 105.05 105.04 107.5 104.91 1.87 Isoflavone Glucoside 96.92 98.25 94.71 96.79 97.57 96.33 96.76 1.25 Ferulic acid 103.83 104.42 103.87 105.87 107.74 103.59 104.89 1.55 <![CDATA[Citrus grandis 'Tomentosa' peel Glycoside > 96.28 97.77 95.6 96.77 96.64 97.38 96.74 0.8 3,6'-Dierucyl Sucrose 96.48 98.85 97.23 99.46 98.84 99.25 98.35 1.23 <![CDATA[Orange peel Glycoside > 96.9 96.78 94.06 95.61 95.69 96.33 95.9 1.09 Nobiletin 101.43 101.77 99.48 98.23 98.37 98.39 99.61 1.61 Tangeretin 94.47 92.48 91.88 92.14 93.81 94.74 93.25 1.33 <![CDATA[ Gao Ben lactone]]> 90.51 90.56 90.15 91.14 90.42 90.49 90.55 0.36 Atractylodes lactone I 96.27 95.37 94.9 94.14 95.47 92.31 94.74 1.46

[0073] Reality

[0074] Example 6

[0075] Limit of Detection and Limit of Quantitation

[0076] The mixed reference solution of Example 1 was gradually diluted, 10 μL was accurately drawn and injected into the liquid chromatograph, and the chromatogram was recorded. The corresponding concentration of the index component to be measured when the signal-to-noise ratio of the chromatogram was 3:1 was taken as the detection limit, and the corresponding concentration of the index component to be measured when the signal-to-noise ratio of the chromatogram was 10:1 was taken as the quantitative limit. After testing, the detection limits of cryptochlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-diesinapoylsucrose, hesperidin, nobiletin, tangeretin, ligustilide and atractylodes lactone I were 0.09, 0.07, 0.10, 0.15, 0.05, 0.08, 0.11, 0.32, 0.09, 0.09, 0.20 and 0.09 μg / mL, respectively, and the quantification limits were 0.22, 0.20, 0.30, 0.31, 0.16, 0.28, 0.33, 0.80, 0.26, 0.21, 0.81 and 0.26 μg / mL, respectively.

[0077] Example 7

[0078] Determination of samples

[0079] According to the method shown in Example 1, 137 production batches of Ginseng Jianpi Pills were tested. The specific test results are shown in Table 9.

[0080] Table 9 Measurement results (n=3, unit: mg / g)

[0081]

[0082] .

Claims

1. A method for simultaneously determining the contents of 12 index components in ginseng Jianpi pills using HPLC wavelength switching technology, characterized in that: HPLC wavelength switching technology was used to determine the cryptochlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-diesinapoylsucrose, hesperidin, nobiletin, tangeretin, ligustilide, and atractylodes lactone I in Renshen Jianpi Pills. The HPLC detection wavelengths were: 0-14 min, 325 nm; 14-20 min, 260 nm; 20-21.5 min, 325 nm; 21.5-22.5 min, 282 nm; 22.5-60 min, 325 nm; 60-70 min, 277 nm; Take 2-3g of Renshen Jianpi Pills, add methanol, heat under reflux in a water bath for 0.5-2h, take out, cool, weigh, make up the lost weight with methanol, shake well, filter, and obtain the test solution; The test solution was injected into HPLC and detected by wavelength switching method. The position and peak area of ​​each chromatographic peak were determined and substituted into the standard curve to calculate the content of each component in the test sample. HPLC chromatographic conditions: chromatographic column: Kromasil 100-5-C 18 , 250 mm×4.6 mm 5 μm, the mobile phase was acetonitrile-0.1% formic acid aqueous solution, gradient elution, 0-5 min, 5% acetonitrile; 5-60 min, 5%→65% acetonitrile; 60-63 min, 65% acetonitrile; 63-66 min, 65%→90% acetonitrile; 66-70 min, 90% acetonitrile; flow rate was 1.0 mL·min -1 ; Column temperature was 35°C; Injection volume was 10 μL.

2. The method for simultaneously determining the contents of 12 index components in Ginseng Jianpi Pills by HPLC wavelength switching technology according to claim 1, characterized in that: The specific steps include: (1) Preparation of mixed reference solution: Weigh chlorogenic acid, vanillic acid, spinosin, calycosin glucoside, ferulic acid, naringin, 3,6'-diesinapoylsucrose, hesperidin, nobiletin, tangeretin, ligustilide, and atractylodes lactone I reference substances respectively, and add methanol to make a solution containing 17.75 μg chlorogenic acid, 20.02 μg vanillic acid, 19.9 μg spinosin, and 10.5 μg atractylodes lactone per 1 mL. A mixed reference solution of 6 μg, 30.81 μg of calycosin glucoside, 21.07 μg of ferulic acid, 84.14 μg of rutin, 44.24 μg of 3,6'-diesinapoylsucrose, 638.38 μg of hesperidin, 17.63 μg of nobiletin, 17.17 μg of tangeretin, 162.48 μg of ligustilide, and 17.28 μg of atractylodes lactone; (2) Drawing of standard curve: Dilute the mixed reference solution into different concentration gradients, inject into HPLC, and detect using wavelength switching method, with mass concentration as the horizontal axis ( X ), the peak area integral value is the ordinate ( Y ), draw the standard curve.

3. The method for simultaneously determining the contents of 12 index components in Ginseng Jianpi Pills by HPLC wavelength switching technology according to claim 1, characterized in that: The ginseng spleen-strengthening pill is one of a water-honey pill, a small honey pill and a large honey pill.

4. The method for simultaneously determining the contents of 12 index components in Ginseng Jianpi Pills by HPLC wavelength switching technology according to claim 3, characterized in that: In step (2), the large honey pills or small honey pills are added with diatomaceous earth and fully ground, then cut into pieces, and then methanol is added. The mass ratio of the large honey pills or small honey pills to the diatomaceous earth is 2-3:1.5.

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