CN112526006A - Method for detecting saikosaponin components in minor bupleurum particles - Google Patents
Method for detecting saikosaponin components in minor bupleurum particles Download PDFInfo
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- CN112526006A CN112526006A CN202011121449.5A CN202011121449A CN112526006A CN 112526006 A CN112526006 A CN 112526006A CN 202011121449 A CN202011121449 A CN 202011121449A CN 112526006 A CN112526006 A CN 112526006A
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- saikosaponin
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- methanol
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- KYWSCMDFVARMPN-LCSVLAELSA-N Saikosaponin D Chemical compound O([C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@]([C@H]3[C@]([C@@H]4[C@@]([C@@]5(C[C@@H](O)[C@]67CO[C@]5([C@@H]6CC(C)(C)CC7)C=C4)C)(C)CC3)(C)CC2)(C)CO)O[C@@H]([C@@H]1O)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O KYWSCMDFVARMPN-LCSVLAELSA-N 0.000 title claims abstract description 57
- 229930192014 saikosaponin Natural products 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 50
- 239000002245 particle Substances 0.000 title claims abstract description 45
- 241000202726 Bupleurum Species 0.000 title description 14
- 239000000243 solution Substances 0.000 claims abstract description 118
- 239000013558 reference substance Substances 0.000 claims abstract description 94
- 241001132254 Bupleurum tenue Species 0.000 claims abstract description 35
- 239000012085 test solution Substances 0.000 claims abstract description 21
- 238000004458 analytical method Methods 0.000 claims abstract description 16
- 238000002360 preparation method Methods 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 231
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 48
- VJEMOEYSQDKAQF-MJKDWHOWSA-N Saikosaponin C Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@@H]2C([C@H]3[C@](C4[C@@]([C@@]5(C[C@H](O)[C@]67CO[C@]5([C@@H]6CC(C)(C)CC7)C=C4)C)(C)CC3)(C)CC2)(C)C)O[C@@H]1CO[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 VJEMOEYSQDKAQF-MJKDWHOWSA-N 0.000 claims description 43
- VSVPCEFIECVNTB-UHFFFAOYSA-N Saikosaponin c Natural products CC1OC(OC2C(O)C(O)C(OC3CCC4(C)C(C3)C(C)(C)CC5(C)C4C=CC67OCC8(CCC(C)(C)CC68)C(O)CC57C)OC2COC9OC(CO)C(O)C(O)C9O)C(O)C(O)C1O VSVPCEFIECVNTB-UHFFFAOYSA-N 0.000 claims description 43
- ZDKCXSMMRXSSDE-UHFFFAOYSA-N chikusakoside II Natural products OC1C(O)C(O)C(C)OC1OC1C(O)C(O)C(OC2C(C3C(C4C(C5(CC(O)C67COC5(C6CC(C)(C)CC7)C=C4)C)(C)CC3)(C)CC2)(C)CO)OC1COC1C(O)C(O)C(O)C(CO)O1 ZDKCXSMMRXSSDE-UHFFFAOYSA-N 0.000 claims description 43
- VJEMOEYSQDKAQF-UHFFFAOYSA-N saikogenin E 3-O-beta-D-glucopyranosyl-(1?6)-[alpha-L-rhamnopyranosyl-(1?4)]-beta-D-glucopyranoside Natural products OC1C(O)C(O)C(C)OC1OC1C(O)C(O)C(OC2C(C3C(C4C(C5(CC(O)C67COC5(C6CC(C)(C)CC7)C=C4)C)(C)CC3)(C)CC2)(C)C)OC1COC1C(O)C(O)C(O)C(CO)O1 VJEMOEYSQDKAQF-UHFFFAOYSA-N 0.000 claims description 43
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- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 claims description 7
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 5
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
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- QLPRYZXNWYTFCI-UHFFFAOYSA-N saikosaponin D Natural products CC1OC(OC2CCC3(C)C(CCC4(C)C3C=CC56OCC7(CCC(C)(C)CC57)C(O)CC46C)C2(C)CO)C(O)C(O)C1OC8OC(CO)C(O)C(O)C8O QLPRYZXNWYTFCI-UHFFFAOYSA-N 0.000 description 22
- PQPVAGWUNWFCJE-UHFFFAOYSA-N saikosaponin a Natural products CC1OC(OC2CCC3(C)C(C2)C(C)(CO)CC4(C)C3C=CC56OCC7(CCC(C)(C)CC57)C(O)CC46C)C(O)C(OC8OC(CO)C(O)C(O)C8O)C1O PQPVAGWUNWFCJE-UHFFFAOYSA-N 0.000 description 22
- KYWSCMDFVARMPN-MSSMMRRTSA-N Saikosaponin A Chemical compound O([C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@]([C@H]3[C@]([C@@H]4[C@@]([C@@]5(C[C@H](O)[C@]67CO[C@]5([C@@H]6CC(C)(C)CC7)C=C4)C)(C)CC3)(C)CC2)(C)CO)O[C@@H]([C@@H]1O)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O KYWSCMDFVARMPN-MSSMMRRTSA-N 0.000 description 19
- WRYJYFCCMSVEPQ-ORAXXRKOSA-N Saikosaponin b2 Chemical compound O([C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@]([C@H]3[C@]([C@@H]4[C@@]([C@@]5(C[C@@H](O)[C@@]6(CO)CCC(C)(C)CC6=C5C=C4)C)(C)CC3)(C)CC2)(C)CO)O[C@@H]([C@@H]1O)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O WRYJYFCCMSVEPQ-ORAXXRKOSA-N 0.000 description 11
- UFEGAVYKHITZAC-RUCQNWDKSA-N (2S,3R,4S,5S,6R)-2-[(2R,3R,4S,5S,6R)-2-[[(3S,4R,4aR,6aS,6bR,8S,8aS,12aS,14bS)-8-hydroxy-4,8a-bis(hydroxymethyl)-4,6a,6b,11,11,14b-hexamethyl-1,2,3,4a,5,6,7,8,9,10,12,12a-dodecahydropicen-3-yl]oxy]-3,5-dihydroxy-6-methyloxan-4-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound C[C@H]1O[C@@H](O[C@H]2CC[C@@]3(C)[C@@H](CC[C@]4(C)C3=CC=C3[C@@H]5CC(C)(C)CC[C@]5(CO)[C@@H](O)C[C@@]43C)[C@]2(C)CO)[C@H](O)[C@@H](O[C@@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@H]1O UFEGAVYKHITZAC-RUCQNWDKSA-N 0.000 description 10
- WRYJYFCCMSVEPQ-MNIDVGFKSA-N (2s,3r,4s,5s,6r)-2-[(2r,3r,4s,5s,6r)-2-[[(3s,4r,4ar,6ar,6bs,8s,8as,14ar,14bs)-8-hydroxy-4,8a-bis(hydroxymethyl)-4,6a,6b,11,11,14b-hexamethyl-1,2,3,4a,5,6,7,8,9,10,12,14a-dodecahydropicen-3-yl]oxy]-3,5-dihydroxy-6-methyloxan-4-yl]oxy-6-(hydroxymethyl)oxane Chemical compound O([C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@]([C@H]3[C@]([C@@H]4[C@@]([C@@]5(C[C@H](O)[C@@]6(CO)CCC(C)(C)CC6=C5C=C4)C)(C)CC3)(C)CC2)(C)CO)O[C@@H]([C@@H]1O)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O WRYJYFCCMSVEPQ-MNIDVGFKSA-N 0.000 description 10
- PYJMYPPFWASOJX-UHFFFAOYSA-N 2-[4,5-dihydroxy-6-[[8-hydroxy-8a-(hydroxymethyl)-4,4,6a,6b,11,11,14b-heptamethyl-1,2,3,4a,5,6,7,8,9,10,12,14a-dodecahydropicen-3-yl]oxy]-2-[[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxymethyl]oxan-3-yl]oxy-6-methyloxane-3,4,5-triol Chemical compound OC1C(O)C(O)C(C)OC1OC1C(O)C(O)C(OC2C(C3C(C4C(C5(CC(O)C6(CO)CCC(C)(C)CC6=C5C=C4)C)(C)CC3)(C)CC2)(C)C)OC1COC1C(O)C(O)C(O)C(CO)O1 PYJMYPPFWASOJX-UHFFFAOYSA-N 0.000 description 10
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention discloses a method for detecting saikosaponin components in bupleurum tenue particles, which comprises the following steps: s1, solution preparation: preparing a mixed reference substance solution and a test solution; s2, performing content analysis by combining a high performance liquid chromatography and an electrospray detector: respectively and precisely sucking 10 mu L and 20 mu L of the mixed reference substance solution prepared in the step S1 and 10-20 mu L of the test sample solution, injecting the mixed reference substance solution into a liquid chromatograph, measuring, and calculating by using an external standard two-point method logarithmic equation; and detecting a chromatographic peak by adopting an electrospray detector. The detection method provided by the invention can meet the inspection requirements of daily samples of traditional Chinese medicine enterprises and provide guarantee for the quality of products.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, in particular to a method for detecting saikosaponin components in small bupleurum particles.
Background
The radix bupleuri granule is derived from classic famous prescription Xiaochaihu decoction of Shanghan Zhang Zhongjing' Shanghai Han miscellaneous diseases Lun, only ginseng is changed into radix codonopsitis, and the radix bupleuri granule is composed of seven medicines of radix bupleuri, radix scutellariae, ginger processed pinellia, radix codonopsitis, ginger, liquorice and Chinese date and is mainly used for relieving exterior syndrome, dissipating heat, soothing liver and harmonizing stomach. In the recipe, chai Hu has the effect of harmonizing Shaoyang, expelling pathogenic factors, soothing liver and relieving depression as the monarch drug. The retail price ratio of the bupleurum tenue granules at the national Chinese patent medicine cold medicine terminal in 2018 is continuously increased to 75.79 percent, which is the absolute leading person of the Chinese patent medicine cold medicines. At present, the bupleurum tenue granules have about 100 approved characters in enterprises, the price of the market is greatly different, and the quality is also uneven.
The Chinese pharmacopoeia (2020 edition) only measures the content of baicalin, does not limit the content of bupleurum serving as a monarch drug in the preparation, and cannot ensure the effectiveness of the preparation. In the quality standard of the similar variety XIAOCHAIHU decoction in the Japanese pharmacopoeia (17 th edition), HPLC method is adopted to measure saikosaponin b2Quantitative control is performed. Chinese patent No. CN 110320286A discloses a method for measuring the content of effective components in XIAOCHAIHU granule, which is to measure the content of baicalin and saikosaponin a in XIAOCHAIHU granule by near infrared spectrum. The traditional Chinese medicine and the compound have the characteristics of integrity, multiple target points and multi-component synergistic effect. In the prescription of Xiaochaihu granules, Bupleurum root, radix bupleuri has the functions of harmonizing Shaoyang, expelling exogenous pathogenic factors, soothing liver and relieving depression, and is the monarch drug. Therefore, the quality of the monarch drug cannot be effectively controlled only by measuring the content of the single index component saikosaponin a in the monarch drug. Chinese patent publication No. CN 103364496B discloses a method for simultaneously determining four saikosaponin (saikosaponin a, saikosaponin c, saikosaponin d, saikosaponin B) in bupleuri radix extract by high performance liquid chromatography-tandem diode array detector (HPLC-PDA) technique2) The method of content. Saikosaponin a, c, d has weak ultraviolet absorption,and saikosaponin b2Has conjugated double bond structure and ultraviolet maximum absorption wavelength of 254nm or so. When the PDA is adopted for detecting wavelength switching, baseline drift is easy to occur, especially for a traditional Chinese medicine compound with complex components, poor separation degree of index components and adjacent chromatographic peaks is easy to cause, and the durability of the method is poor. In addition, for compounds with weak ultraviolet absorption of saikosaponin a, c, d, the detection sensitivity of UV or PDA is poor. Determining contents [ J ] of saikosaponin a, b1, b2, c in bupleuri radix granule simultaneously by HPLC-MS]In 2016(23) and 2068-2071), high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to simultaneously determine four saikosaponin (saikosaponin a and saikosaponin b) in bupleurum formula granule1Saikosaponin d and saikosaponin b2) The content of (a). LC-MS-MS research of saikosaponin A and saikosaponin D in certain Chinese medicinal compound [ J]The analysis and test bulletin, 2001,20(20):71-72) and the like, studies the transformation of the saikosaponin components in the Chinese medicinal preparation containing bupleurum. Researches show that the saikosaponin a and the saikosaponin d are easy to be rearranged and converted into the saikosaponin b with a conjugated double bond structure under the acidic condition or the heating condition1And b2. High resolution mass spectrometry (LC-Q active) is adopted in the early stageTM) Qualitative analysis of the granule of Bupleurum tenue will not detect saikoside d, indicating that saikoside d has been completely converted into saikoside b2. The mass spectrometer has high price, high maintenance cost and low popularization degree, cannot meet the daily sample inspection requirement of a traditional Chinese medicine enterprise, and has poor applicability. Therefore, the above patent and analysis methods are not completely suitable for quality control of Xiao chai Hu granules. The existing quality standard and analysis and detection method do not effectively control the monarch drug bupleurum in the formula of the bupleurum tenue, so that the quality of the product cannot be ensured.
Electrospray detectors (CAD) are a new general type of mass detector. It is independent of the molecular structure of the compound, and can detect most of non-volatile and semi-volatile compounds, especially compounds without chromophoric groups. Compared with an Evaporative Light Scattering Detector (ELSD), CAD has the characteristics of high sensitivity, good reproducibility and stability, wide dynamic detection range and the like, is simple to operate and convenient to maintain, and is one of ideal detection means in the field of drug analysis.
Therefore, it is urgent to establish a simple, accurate and practical method for simultaneously measuring saikosaponin components in small bupleurum particles, so as to effectively and feasibly control the product quality.
Disclosure of Invention
The invention aims to provide a method for detecting saikosaponin components in bupleurum tenue particles aiming at the defects in the prior art, which can meet the inspection requirements of daily samples of traditional Chinese medicine enterprises and provide guarantee for the quality of products.
In order to achieve the purpose, the invention adopts the technical scheme that:
provides a method for detecting saikosaponin components in bupleurum tenue particles, which comprises the following steps:
s1, solution preparation:
preparing a reference substance solution: collecting saikosaponin C (SSc), I (SSi), H (SSh), A (SSa), B2(SSb2)、G(SSg)、B1(SSb1) Accurately weighing appropriate amount of reference substance, respectively placing in volumetric flasks, adding methanol for dissolving, and preparing into reference substance solution with required concentration;
preparing a mixed reference substance solution: precisely measuring the 7 reference substance solutions, and preparing a mixed reference substance solution with the required mass concentration for later use;
preparing a test solution: grinding proper amount of small radix bupleuri particles, precisely weighing, adding water to completely dissolve, performing activated C18 solid phase extraction on a small column, sequentially eluting with 10% methanol solution containing 5% concentrated ammonia or 1% NaOH, 30% methanol and analytically pure methanol, collecting methanol eluate, evaporating to dryness, dissolving residue with methanol, fixing volume to a volumetric flask, and filtering with a microporous membrane to obtain a test solution;
s2, performing content analysis by combining a high performance liquid chromatography and an electrospray detector:
respectively and precisely sucking the mixed reference solution and the test solution prepared in the step S1, injecting the mixed reference solution and the test solution into a liquid chromatograph, measuring, and calculating by using an external standard two-point method logarithmic equation; detecting a chromatographic peak by adopting an electrospray detector;
chromatographic conditions are as follows: mobile phase A: an aqueous solution containing 0.01% acetic acid or 0.01% formic acid, a 10% acetonitrile-90% aqueous solution (containing 0.1mmol of ammonium acetate, pH 4.0); mobile phase B: chromatographically purifying acetonitrile, 90% acetonitrile-10% water solution (containing 0.1mmol ammonium acetate, pH 4.0), and gradient eluting; chromatography column Waters CORTECTS C18 or chromatography column Thermo Hypersil GOLD; column temperature: 25-35 ℃; flow rate: 0.7-0.9 mL/min;
electrospray detector conditions: electrospray detector, nitrogen pressure: 35-50psi, span: 100pA, electrospray temperature: at 50 ℃.
Preferably, in S1, the C18 solid phase extraction cartridge is pre-washed with 6mL of methanol and then equilibrated with 10mL of water.
Preferably, in S2, the chromatographic column Waters CORTECTS C18 has a column length of 15cm, an inner diameter of 4.6mm, and a filler particle size of 2.7 μm.
Preferably, in S2, the chromatographic column Thermo Hypersil GOLD has a column length of 15cm, an inner diameter of 4.6mm, and a filler particle size of 3 μm.
Preferably, the chromatography column Waters CORTECTS C18 or chromatography column Thermo Hypersil GOLD uses octadecylsilane bonded silica gel as a packing agent.
Preferably, when the mobile phase A adopts an aqueous solution containing 0.01% of acetic acid or 0.01% of formic acid, and the mobile phase B adopts chromatographic pure acetonitrile, the elution gradient is as follows:
0～55min，30～32％B；
55～58min，32～90％B；
58～61min，90～30％B；
61～70min，30％B。
preferably, when the mobile phase A uses a 10% acetonitrile aqueous solution containing 0.1mmol of ammonium acetate (pH 4.0) and the mobile phase B uses a 90% acetonitrile aqueous solution containing 0.1mmol of ammonium acetate (pH 4.0), the elution gradient is:
0～35min，25～35％B；
35～37min，35～90％B；
37～45min，90～25％B；
45～50min，25％B。
by adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
1. the invention adopts the high performance liquid chromatography-electrospray detector combined technology (HPLC-CAD) to simultaneously determine the content of 7 saikosaponin in the bupleurum tenue particles, and the method is simple, reliable, good in durability and advanced.
2. The method is adopted to determine the content of saikosaponin in 15 batches of small bupleurum particles sold in the market, and the result shows that the method can be used for evaluating the consistency of the small bupleurum products among batches and the controllability of the process. The invention establishes a method for measuring the content of multicomponent saikoside, can comprehensively control the quality of bupleurum tenue particles and provides reference for improving the quality of the bupleurum tenue particles.
Drawings
FIG. 1 shows a typical chromatogram for HPLC-CAD according to the present invention (wherein: A-blank solvent; B-Bupleurum-deficient negative preparation; C-mixed reference solution; D-test solution; 1-SSc; 2-SSi; 3-SSh; 4-SSa; 5-SSb)1；6-SSg；7-SSb2)。
Detailed Description
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
Example 1
A method for detecting saikosaponin components in bupleurum tenue particles comprises the following steps:
s1, solution preparation:
preparing a reference substance solution: collecting saikosaponin C (SSc), I (SSi), H (SSh), A (SSa), B2(SSb2)、G(SSg)、B1(SSb1) Accurately weighing appropriate amount of reference substance, respectively placing in volumetric flasks, adding methanol for dissolving, and preparing into reference substance solution with required concentration;
preparing a mixed reference substance solution: precision (precision)Measuring the 7 reference substance solutions to prepare a mixed reference substance solution with the required mass concentration, namely each l of the mixed reference substance solution contains 10 mu g of SSc, 10 mu g of SSi, 10 mu g of SSh, 30 mu g of SSa and 30 mu g of SSb2100μg、SSg 20μg、SSb1Taking 50 mu g of solution as a mixed reference solution for later use;
preparing a test solution: proper amount of bupleurum tenue particles are ground into fine powder, 3g is precisely weighed, 6mL of water is added to be completely dissolved, the active Waters Sep-Pak C18 solid phase extraction column is used (firstly, 6mL of methanol is used for prewashing, then, 10mL of water is used for balancing), 10mL of methanol solution containing 5% concentrated ammonia, 30% of methanol and 10mL of methanol are used for elution in sequence, methanol eluent is collected and evaporated to dryness, residues are dissolved by methanol, the volume is determined to be 2mL, and a 0.22 mu m microporous membrane is used for filtration, thus obtaining a sample solution;
s2, performing content analysis by combining a high performance liquid chromatography and an electrospray detector:
respectively and precisely sucking 10 mu L and 20 mu L of the mixed reference substance solution and 10 mu L of the test sample solution, injecting the mixed reference substance solution and the test sample solution into a liquid chromatograph, measuring, and calculating by using an external standard two-point method logarithmic equation; detecting a chromatographic peak by adopting an electrospray detector;
chromatographic conditions are as follows: mobile phase A: 0.01% acetic acid in water, mobile phase B: chromatographically pure acetonitrile, gradient elution: 0-55 min, 30-32% B; 55-58 min, 32-90% B; 58-61 min, 90-30% B; 61-70 min, 30% B; chromatography column Waters CORTECTS C18 (4.6X 150mm, 2.7 μm); column temperature: 30 ℃; flow rate: 0.8 mL/min;
electrospray detector conditions: electrospray detector, nitrogen pressure: 50psi, range: 100pA, electrospray temperature: at 50 ℃.
The content of saikosaponin in the granule is such that each bag contains saikosaponin C (C)48H78O17) Saikosaponin I (C)48H78O17) And saikosaponin H (C)48H78O17) And saikosaponin A (C)42H68O13) And saikosaponin B2(C42H68O13) Saikosaponin G (C)42H68O13) And saikosaponin B1(C42H68O13) The total amount of (c) is calculated.
(1) Standard curve:
5 mixed control solutions of different concentrations were prepared, 10. mu.L of each solution was pipetted and injected into a liquid chromatograph. And taking the log C of the concentration of the reference solution as an abscissa x, and taking the log A of the peak area as an ordinate y to perform linear regression. The results are shown in table 1:
TABLE 1 Linear regression equation and Linear Range
As can be seen from the data in Table 1, 7 saikosaponin showed good linear relationship in each linear range.
(2) Precision:
taking the mixed reference substance solution, continuously sampling for 6 times, continuously sampling for 3 days, measuring peak areas of 7 saikosaponin, calculating relative standard deviation (RSD%), and inspecting precision within day and during day. The result shows that the RSD percent of the precision of the internal day and the daytime is not more than 2.1 percent, and the method has good precision.
(3) Repeatability:
weighing 6 parts of the same batch of small Bupleurum particles, preparing a test solution, injecting a sample, and measuring the peak areas of 7 saikosaponin, namely SSc, SSi, SSh, SSa and SSb2SSg and SSb1The relative standard deviations (RSD%) were 5.2%, 6.0%, 6.8%, 3.6%, 4.4%, 5.3%, respectively (n ═ 6), indicating good reproducibility of the method.
(4) Accuracy:
weighing 9 parts of small bupleurum particles with known content, adding each reference substance with the amount equivalent to the concentration levels of 50%, 100% and 150% of each saikosaponin content in the small bupleurum particles, wherein each concentration is 3 parts in parallel. Preparing a test solution, injecting a sample, and measuring the peak area. Calculating the content of each component by a standard curve method, and calculating the recovery rate. The results are shown in table 2:
TABLE 2 recovery results
Application example
Determination of 15 batches of samples:
taking 15 batches of small bupleuri radix granules (produced by Guangzhou Baiyunshan Guanghua pharmacy Co., Ltd., batch number: K90106, K90108, K90116, K90117, K90125, K90141, K90143, K90148, K90150, K90155, K90156, K90157, K90158, K90159 and K90162), and determining according to the above method, wherein the content of saikosaponin in the small bupleuri radix granules is determined by that each bag contains saikosaponin C (C90157)48H78O17) Saikosaponin I (C)48H78O17) And saikosaponin H (C)48H78O17) And saikosaponin A (C)42H68O13) And saikosaponin B2(C42H68O13) Saikosaponin G (C)42H68O13) And saikosaponin B1(C42H68O13) The content of 15 samples is more than 1.25mg in total amount.
Example 2
A method for detecting saikosaponin components in bupleurum tenue particles comprises the following steps:
s1, solution preparation:
preparing a reference substance solution: collecting saikosaponin C (SSc), I (SSi), H (SSh), A (SSa), B2(SSb2)、G(SSg)、B1(SSb1) Accurately weighing appropriate amount of reference substance, respectively placing in volumetric flasks, adding methanol for dissolving, and preparing into reference substance solution with required concentration;
preparing a mixed reference substance solution: precisely measuring the 7 contrastsThe sample solution is prepared into a mixed reference solution with the required mass concentration, namely each liter of the mixed reference solution contains 10 mu g of SSc, 10 mu g of SSi, 10 mu g of SSh, 30 mu g of SSa and SSb2100μg、SSg 20μg、SSb1Taking 50 mu g of solution as a mixed reference solution for later use;
preparing a test solution: proper amount of bupleurum tenue particles are ground into fine powder, 3g is precisely weighed, 6mL of water is added to be completely dissolved, the active Waters Sep-Pak C18 solid phase extraction column is used (firstly, 6mL of methanol is used for prewashing, then, 10mL of water is used for balancing), 10mL of methanol solution containing 5% concentrated ammonia, 30% of methanol and 10mL of methanol are used for elution in sequence, methanol eluent is collected and evaporated to dryness, residues are dissolved by methanol, the volume is determined to be 2mL, and a 0.22 mu m microporous membrane is used for filtration, thus obtaining a sample solution;
s2, performing content analysis by combining a high performance liquid chromatography and an electrospray detector:
respectively and precisely sucking 10 mu L and 20 mu L of the mixed reference substance solution and 10 mu L of the test sample solution, injecting the mixed reference substance solution and the test sample solution into a liquid chromatograph, measuring, and calculating by using an external standard two-point method logarithmic equation; detecting a chromatographic peak by adopting an electrospray detector;
chromatographic conditions are as follows: mobile phase A: 0.01% acetic acid in water, mobile phase B: chromatographically pure acetonitrile, gradient elution: 0-55 min, 30-32% B; 55-58 min, 32-90% B; 58-61 min, 90-30% B; 61-70 min, 30% B; chromatography column Waters CORTECTS C18 (4.6X 150mm, 2.7 μm); column temperature: 30 ℃; flow rate: 0.7 mL/min;
electrospray detector conditions: electrospray detector, nitrogen pressure: 50psi, range: 100pA, electrospray temperature: at 50 ℃.
The content of saikosaponin in the granule is such that each bag contains saikosaponin C (C)48H78O17) Saikosaponin I (C)48H78O17) And saikosaponin H (C)48H78O17) And saikosaponin A (C)42H68O13) And saikosaponin B2(C42H68O13) Saikosaponin G (C)42H68O13) And saikosaponin B1(C42H68O13) The total amount was 1.63 mg.
Example 3
A method for detecting saikosaponin components in bupleurum tenue particles comprises the following steps:
s1, solution preparation:
preparing a reference substance solution: collecting saikosaponin C (SSc), I (SSi), H (SSh), A (SSa), B2(SSb2)、G(SSg)、B1(SSb1) Accurately weighing appropriate amount of reference substance, respectively placing in volumetric flasks, adding methanol for dissolving, and preparing into reference substance solution with required concentration;
preparing a mixed reference substance solution: precisely measuring the 7 reference substance solutions to prepare a mixed reference substance solution with the required mass concentration, namely each l of the mixed reference substance solution contains 10 mu g of SSc, 10 mu g of SSi, 10 mu g of SSh, 30 mu g of SSa and 30 mu g of SSb2100μg、SSg 20μg、SSb1Taking 50 mu g of solution as a mixed reference solution for later use;
preparing a test solution: proper amount of bupleurum tenue particles are ground into fine powder, 3g is precisely weighed, 6mL of water is added to be completely dissolved, the active Waters Sep-Pak C18 solid phase extraction column is used (firstly, 6mL of methanol is used for prewashing, then, 10mL of water is used for balancing), 10mL of methanol solution containing 5% concentrated ammonia, 30% of methanol and 10mL of methanol are used for elution in sequence, methanol eluent is collected and evaporated to dryness, residues are dissolved by methanol, the volume is determined to be 2mL, and a 0.22 mu m microporous membrane is used for filtration, thus obtaining a sample solution;
s2, performing content analysis by combining a high performance liquid chromatography and an electrospray detector:
respectively and precisely sucking 10 mu L and 20 mu L of the mixed reference substance solution and 10 mu L of the test sample solution, injecting the mixed reference substance solution and the test sample solution into a liquid chromatograph, measuring, and calculating by using an external standard two-point method logarithmic equation; detecting a chromatographic peak by adopting an electrospray detector;
chromatographic conditions are as follows: mobile phase A: 0.01% acetic acid in water, mobile phase B: chromatographically pure acetonitrile, gradient elution: 0-55 min, 30-32% B; 55-58 min, 32-90% B; 58-61 min, 90-30% B; 61-70 min, 30% B; chromatography column Waters CORTECTS C18 (4.6X 150mm, 2.7 μm); column temperature: 30 ℃; flow rate: 0.9 mL/min;
electrospray detector conditions: electrospray detector, nitrogen pressure: 50psi, range: 100pA, electrospray temperature: at 50 ℃.
The content of saikosaponin in the granule is such that each bag contains saikosaponin C (C)48H78O17) Saikosaponin I (C)48H78O17) And saikosaponin H (C)48H78O17) And saikosaponin A (C)42H68O13) And saikosaponin B2(C42H68O13) Saikosaponin G (C)42H68O13) And saikosaponin B1(C42H68O13) The total amount was 1.50 mg.
Example 4
A method for detecting saikosaponin components in bupleurum tenue particles comprises the following steps:
s1, solution preparation:
preparing a reference substance solution: collecting saikosaponin C (SSc), I (SSi), H (SSh), A (SSa), B2(SSb2)、G(SSg)、B1(SSb1) Accurately weighing appropriate amount of reference substance, respectively placing in volumetric flasks, adding methanol for dissolving, and preparing into reference substance solution with required concentration;
preparing a mixed reference substance solution: precisely measuring the 7 reference substance solutions to prepare a mixed reference substance solution with the required mass concentration, namely each l of the mixed reference substance solution contains 10 mu g of SSc, 10 mu g of SSi, 10 mu g of SSh, 30 mu g of SSa and 30 mu g of SSb2100μg、SSg 20μg、SSb1Taking 50 mu g of solution as a mixed reference solution for later use;
preparing a test solution: proper amount of bupleurum tenue particles are ground into fine powder, 3g is precisely weighed, 6mL of water is added to be completely dissolved, the active Waters Sep-Pak C18 solid phase extraction column is used (firstly, 6mL of methanol is used for prewashing, then, 10mL of water is used for balancing), 10mL of methanol solution containing 5% concentrated ammonia, 30% of methanol and 10mL of methanol are used for elution in sequence, methanol eluent is collected and evaporated to dryness, residues are dissolved by methanol, the volume is determined to be 2mL, and a 0.22 mu m microporous membrane is used for filtration, thus obtaining a sample solution;
s2, performing content analysis by combining a high performance liquid chromatography and an electrospray detector:
respectively and precisely sucking 10 mu L and 20 mu L of the mixed reference substance solution and 10 mu L of the test sample solution, injecting the mixed reference substance solution and the test sample solution into a liquid chromatograph, measuring, and calculating by using an external standard two-point method logarithmic equation; detecting a chromatographic peak by adopting an electrospray detector;
chromatographic conditions are as follows: mobile phase A: 0.01% acetic acid in water, mobile phase B: chromatographically pure acetonitrile, gradient elution: 0-55 min, 30-32% B; 55-58 min, 32-90% B; 58-61 min, 90-30% B; 61-70 min, 30% B; chromatography column Waters CORTECTS C18 (4.6X 150mm, 2.7 μm); column temperature: 25 ℃; flow rate: 0.8 mL/min;
electrospray detector conditions: electrospray detector, nitrogen pressure: 50psi, range: 100pA, electrospray temperature: at 50 ℃.
The content of saikosaponin in the granule is such that each bag contains saikosaponin C (C)48H78O17) Saikosaponin I (C)48H78O17) And saikosaponin H (C)48H78O17) And saikosaponin A (C)42H68O13) And saikosaponin B2(C42H68O13) Saikosaponin G (C)42H68O13) And saikosaponin B1(C42H68O13) The total amount was 1.52 mg.
Example 5
A method for detecting saikosaponin components in bupleurum tenue particles comprises the following steps:
s1, solution preparation:
preparing a reference substance solution: collecting saikosaponin C (SSc), I (SSi), H (SSh), A (SSa), B2(SSb2)、G(SSg)、B1(SSb1) Accurately weighing appropriate amount of reference substance, respectively placing in volumetric flasks, adding methanol for dissolving, and preparing into reference substance solution with required concentration;
preparing a mixed reference substance solution: precisely measuring the 7 reference substance solutions to prepare a mixed reference substance solution with required mass concentrationI.e., 10. mu.g SSc, 10. mu.g SSi, 10. mu.g SSh, 30. mu.g SSa, SSb per mL2100μg、SSg 20μg、SSb1Taking 50 mu g of solution as a mixed reference solution for later use;
preparing a test solution: proper amount of bupleurum tenue particles are ground into fine powder, 3g is precisely weighed, 6mL of water is added to be completely dissolved, the active Waters Sep-Pak C18 solid phase extraction column is used (firstly, 6mL of methanol is used for prewashing, then, 10mL of water is used for balancing), 10mL of methanol solution containing 5% concentrated ammonia, 30% of methanol and 10mL of methanol are used for elution in sequence, methanol eluent is collected and evaporated to dryness, residues are dissolved by methanol, the volume is determined to be 2mL, and a 0.22 mu m microporous membrane is used for filtration, thus obtaining a sample solution;
s2, performing content analysis by combining a high performance liquid chromatography and an electrospray detector:
respectively and precisely sucking 10 mu L and 20 mu L of the mixed reference substance solution and 10 mu L of the test sample solution, injecting the mixed reference substance solution and the test sample solution into a liquid chromatograph, measuring, and calculating by using an external standard two-point method logarithmic equation; detecting a chromatographic peak by adopting an electrospray detector;
chromatographic conditions are as follows: mobile phase A: 0.01% acetic acid in water, mobile phase B: chromatographically pure acetonitrile, gradient elution: 0-55 min, 30-32% B; 55-58 min, 32-90% B; 58-61 min, 90-30% B; 61-70 min, 30% B; chromatography column Waters CORTECTS C18 (4.6X 150mm, 2.7 μm); column temperature: 35 ℃; flow rate: 0.8 mL/min;
electrospray detector conditions: electrospray detector, nitrogen pressure: 50psi, range: 100pA, electrospray temperature: at 50 ℃.
The content of saikosaponin in the granule is such that each bag contains saikosaponin C (C)48H78O17) Saikosaponin I (C)48H78O17) And saikosaponin H (C)48H78O17) And saikosaponin A (C)42H68O13) And saikosaponin B2(C42H68O13) Saikosaponin G (C)42H68O13) And saikosaponin B1(C42H68O13) The total amount was 1.53 mg.
Example 6
A method for detecting saikosaponin components in bupleurum tenue particles comprises the following steps:
s1, solution preparation:
preparing a reference substance solution: collecting saikosaponin C (SSc), I (SSi), H (SSh), A (SSa), B2(SSb2)、G(SSg)、B1(SSb1) Accurately weighing appropriate amount of reference substance, respectively placing in volumetric flasks, adding methanol for dissolving, and preparing into reference substance solution with required concentration;
preparing a mixed reference substance solution: precisely measuring the 7 reference substance solutions to prepare a mixed reference substance solution with the required mass concentration, namely each l of the mixed reference substance solution contains 10 mu g of SSc, 10 mu g of SSi, 10 mu g of SSh, 30 mu g of SSa and 30 mu g of SSb2100μg、SSg 20μg、SSb1Taking 50 mu g of solution as a mixed reference solution for later use;
preparing a test solution: proper amount of bupleurum tenue particles are ground into fine powder, 3g is precisely weighed, 6mL of water is added to be completely dissolved, the active Waters Sep-Pak C18 solid phase extraction column is used (firstly, 6mL of methanol is used for prewashing, then, 10mL of water is used for balancing), 10mL of methanol solution containing 5% concentrated ammonia, 30% of methanol and 10mL of methanol are used for elution in sequence, methanol eluent is collected and evaporated to dryness, residues are dissolved by methanol, the volume is determined to be 2mL, and a 0.22 mu m microporous membrane is used for filtration, thus obtaining a sample solution;
s2, performing content analysis by combining a high performance liquid chromatography and an electrospray detector:
respectively and precisely sucking 10 mu L and 20 mu L of the mixed reference substance solution and 10 mu L of the test sample solution, injecting the mixed reference substance solution and the test sample solution into a liquid chromatograph, measuring, and calculating by using an external standard two-point method logarithmic equation; detecting a chromatographic peak by adopting an electrospray detector;
chromatographic conditions are as follows: mobile phase A: 0.01% acetic acid in water, mobile phase B: chromatographically pure acetonitrile, gradient elution: 0-55 min, 30-32% B; 55-58 min, 32-90% B; 58-61 min, 90-30% B; 61-70 min, 30% B; chromatography column Waters CORTECTS C18 (4.6X 150mm, 2.7 μm); column temperature: 30 ℃; flow rate: 0.8 mL/min;
electrospray detector conditions: electrospray detector, nitrogen pressure: 35psi, span: 100pA, electrospray temperature: at 50 ℃.
The content of saikosaponin in the granule is such that each bag contains saikosaponin C (C)48H78O17) Saikosaponin I (C)48H78O17) And saikosaponin H (C)48H78O17) And saikosaponin A (C)42H68O13) And saikosaponin B2(C42H68O13) Saikosaponin G (C)42H68O13) And saikosaponin B1(C42H68O13) The total amount was 1.65 mg.
Example 7
A method for detecting saikosaponin components in bupleurum tenue particles comprises the following steps:
s1, solution preparation:
preparing a reference substance solution: collecting saikosaponin C (SSc), I (SSi), H (SSh), A (SSa), B2(SSb2)、G(SSg)、B1(SSb1) Accurately weighing appropriate amount of reference substance, respectively placing in volumetric flasks, adding methanol for dissolving, and preparing into reference substance solution with required concentration;
preparing a mixed reference substance solution: precisely measuring the 7 reference substance solutions to prepare a mixed reference substance solution with the required mass concentration, namely each l of the mixed reference substance solution contains 10 mu g of SSc, 10 mu g of SSi, 10 mu g of SSh, 30 mu g of SSa and 30 mu g of SSb2100μg、SSg 20μg、SSb1Taking 50 mu g of solution as a mixed reference solution for later use;
preparing a test solution: proper amount of bupleurum tenue particles are ground into fine powder, 3g is precisely weighed, 6mL of water is added to be completely dissolved, the active Waters Sep-Pak C18 solid phase extraction column is used (firstly, 6mL of methanol is used for prewashing, then, 10mL of water is used for balancing), 10mL of methanol solution containing 5% concentrated ammonia, 30% of methanol and 10mL of methanol are used for elution in sequence, methanol eluent is collected and evaporated to dryness, residues are dissolved by methanol, the volume is determined to be 2mL, and a 0.22 mu m microporous membrane is used for filtration, thus obtaining a sample solution;
s2, performing content analysis by combining a high performance liquid chromatography and an electrospray detector:
respectively and precisely sucking 10 mu L and 20 mu L of the mixed reference substance solution and 10 mu L of the test sample solution, injecting the mixed reference substance solution and the test sample solution into a liquid chromatograph, measuring, and calculating by using an external standard two-point method logarithmic equation; detecting a chromatographic peak by adopting an electrospray detector;
chromatographic conditions are as follows: mobile phase A: aqueous solution containing 0.01% formic acid, mobile phase B: chromatographically pure acetonitrile, gradient elution: 0-55 min, 30-32% B; 55-58 min, 32-90% B; 58-61 min, 90-30% B; 61-70 min, 30% B; chromatography column Waters CORTECTS C18 (4.6X 150mm, 2.7 μm); column temperature: 30 ℃; flow rate: 0.8 mL/min;
electrospray detector conditions: electrospray detector, nitrogen pressure: 50psi, range: 100pA, electrospray temperature: at 50 ℃.
The content of saikosaponin in the granule is such that each bag contains saikosaponin C (C)48H78O17) Saikosaponin I (C)48H78O17) And saikosaponin H (C)48H78O17) And saikosaponin A (C)42H68O13) And saikosaponin B2(C42H68O13) Saikosaponin G (C)42H68O13) And saikosaponin B1(C42H68O13) The total amount was 1.68 mg.
Example 8
A method for detecting saikosaponin components in bupleurum tenue particles comprises the following steps:
s1, solution preparation:
preparing a reference substance solution: collecting saikosaponin C (SSc), I (SSi), H (SSh), A (SSa), B2(SSb2)、G(SSg)、B1(SSb1) Accurately weighing appropriate amount of reference substance, respectively placing in volumetric flasks, adding methanol for dissolving, and preparing into reference substance solution with required concentration;
preparing a mixed reference substance solution: precisely measuring the 7 reference substance solutions to prepare a mixed reference substance solution with the required mass concentration, namely each liter of the mixed reference substance solution contains 10 mu g of SSc, 10 mu g of SSi, 10 mu g of SSh, 30 mu g of SSa,SSb2100μg、SSg 20μg、SSb1Taking 50 mu g of solution as a mixed reference solution for later use;
preparing a test solution: proper amount of bupleurum tenue particles are ground into fine powder, 3g is precisely weighed, 6mL of water is added to be completely dissolved, the active Waters Sep-Pak C18 solid phase extraction column is used (firstly, 6mL of methanol is used for prewashing, then, 10mL of water is used for balancing), 10mL of methanol solution containing 5% concentrated ammonia, 30% of methanol and 10mL of methanol are used for elution in sequence, methanol eluent is collected and evaporated to dryness, residues are dissolved by methanol, the volume is determined to be 2mL, and a 0.22 mu m microporous membrane is used for filtration, thus obtaining a sample solution;
s2, performing content analysis by combining a high performance liquid chromatography and an electrospray detector:
respectively and precisely sucking 10 mu L and 20 mu L of the mixed reference substance solution and 10 mu L of the test sample solution, injecting the mixed reference substance solution and the test sample solution into a liquid chromatograph, measuring, and calculating by using an external standard two-point method logarithmic equation; detecting a chromatographic peak by adopting an electrospray detector;
chromatographic conditions are as follows: mobile phase A: 10% acetonitrile-90% water (containing 0.1mmol ammonium acetate, pH 4.0), mobile phase B: 90% acetonitrile-10% water (containing 0.1mmol ammonium acetate, pH 4.0), gradient elution was performed: 0-35 min, 25-35% B; 35-37 min, 35-90% B; 37-45 min, 90-25% B; 45-50 min, 25% B; column Thermo Hypersil GOLD (4.6X 150mm, 3 μm); column temperature: 30 ℃; flow rate: 0.8 mL/min;
electrospray detector conditions: electrospray detector, nitrogen pressure: 50psi, range: 100pA, electrospray temperature: at 50 ℃.
The content of saikosaponin in the granule is such that each bag contains saikosaponin C (C)48H78O17) Saikosaponin I (C)48H78O17) And saikosaponin H (C)48H78O17) And saikosaponin A (C)42H68O13) And saikosaponin B2(C42H68O13) Saikosaponin G (C)42H68O13) And saikosaponin B1(C42H68O13) The total amount was 1.58 mg.
Example 9
A method for detecting saikosaponin components in bupleurum tenue particles comprises the following steps:
s1, solution preparation:
preparing a reference substance solution: collecting saikosaponin C (SSc), I (SSi), H (SSh), A (SSa), B2(SSb2)、G(SSg)、B1(SSb1) Accurately weighing appropriate amount of reference substance, respectively placing in volumetric flasks, adding methanol for dissolving, and preparing into reference substance solution with required concentration;
preparing a mixed reference substance solution: precisely measuring the 7 reference substance solutions to prepare a mixed reference substance solution with the required mass concentration, namely each l of the mixed reference substance solution contains 10 mu g of SSc, 10 mu g of SSi, 10 mu g of SSh, 30 mu g of SSa and 30 mu g of SSb2100μg、SSg 20μg、SSb1Taking 50 mu g of solution as a mixed reference solution for later use;
preparing a test solution: proper amount of bupleurum tenue particles are ground into fine powder, 3g is precisely weighed, 6mL of water is added to be completely dissolved, the active Waters Sep-Pak C18 solid phase extraction column is used (firstly, 6mL of methanol is used for prewashing, then, 10mL of water is used for balancing), 10mL of 10% methanol solution containing 1% NaOH, 30% methanol and 10mL of methanol are used for elution in sequence, methanol eluent is collected and evaporated to dryness, residues are dissolved by methanol, the volume is determined to be 2mL volumetric flask, and a 0.22 mu m microporous membrane is used for filtration, thus obtaining a sample solution;
s2, performing content analysis by combining a high performance liquid chromatography and an electrospray detector:
respectively and precisely sucking 10 mu L and 20 mu L of the mixed reference substance solution and 10 mu L of the test sample solution, injecting the mixed reference substance solution and the test sample solution into a liquid chromatograph, measuring, and calculating by using an external standard two-point method logarithmic equation; detecting a chromatographic peak by adopting an electrospray detector;
chromatographic conditions are as follows: mobile phase A: 0.01% acetic acid in water, mobile phase B: chromatographically pure acetonitrile, gradient elution: 0-55 min, 30-32% B; 55-58 min, 32-90% B; 58-61 min, 90-30% B; 61-70 min, 30% B; chromatography column Waters CORTECTS C18 (4.6X 150mm, 2.7 μm); column temperature: 30 ℃; flow rate: 0.8 mL/min;
electrospray detector conditions: electrospray detector, nitrogen pressure: 50psi, range: 100pA, electrospray temperature: at 50 ℃.
The content of saikosaponin in the granule is such that each bag contains saikosaponin C (C)48H78O17) Saikosaponin I (C)48H78O17) And saikosaponin H (C)48H78O17) And saikosaponin A (C)42H68O13) And saikosaponin B2(C42H68O13) Saikosaponin G (C)42H68O13) And saikosaponin B1(C42H68O13) The total amount was 1.60 mg.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Claims (7)
1. A method for detecting saikosaponin components in bupleurum tenue particles is characterized by comprising the following steps:
s1, solution preparation:
preparing a reference substance solution: collecting saikosaponin C (SSc), I (SSi), H (SSh), A (SSa), B2(SSb2)、G(SSg)、B1(SSb1) Accurately weighing appropriate amount of reference substance, respectively placing in volumetric flasks, adding methanol for dissolving, and preparing into reference substance solution with required concentration;
preparing a mixed reference substance solution: precisely measuring the 7 reference substance solutions, and preparing a mixed reference substance solution with the required mass concentration for later use;
preparing a test solution: grinding proper amount of small radix bupleuri particles, precisely weighing, adding water to completely dissolve, performing activated C18 solid phase extraction on a small column, sequentially eluting with 10% methanol solution containing 5% concentrated ammonia or 1% NaOH, 30% methanol and analytically pure methanol, collecting methanol eluate, evaporating to dryness, dissolving residue with methanol, fixing volume to a volumetric flask, and filtering with a microporous membrane to obtain a test solution;
s2, performing content analysis by combining a high performance liquid chromatography and an electrospray detector:
respectively and precisely sucking the mixed reference solution and the test solution prepared in the step S1, injecting the mixed reference solution and the test solution into a liquid chromatograph, measuring, and calculating by using an external standard two-point method logarithmic equation; detecting a chromatographic peak by adopting an electrospray detector;
chromatographic conditions are as follows: mobile phase A: an aqueous solution containing 0.01% acetic acid or 0.01% formic acid, a 10% acetonitrile-90% aqueous solution (containing 0.1mmol of ammonium acetate, pH 4.0); mobile phase B: chromatographically purifying acetonitrile, 90% acetonitrile-10% water solution (containing 0.1mmol ammonium acetate, pH 4.0), and gradient eluting; chromatography column Waters CORTECTS C18 or chromatography column Thermo Hypersil GOLD; column temperature: 25-35 ℃; flow rate: 0.7-0.9 mL/min;
electrospray detector conditions: electrospray detector, nitrogen pressure: 35-50psi, span: 100pA, electrospray temperature: at 50 ℃.
2. The method for detecting the saikosaponin components in the bupleurum tenue particles as claimed in claim 1, wherein in S1, the C18 solid phase extraction cartridge is pre-washed with 6mL of methanol and then equilibrated with 10mL of water.
3. The method for detecting the saikosaponin components in the bupleurum tenue particles as claimed in claim 1, wherein in S2, the chromatographic column Waters CORTECTS C18 has a column length of 15cm, an inner diameter of 4.6mm, and a filler particle size of 2.7 μm.
4. The method for detecting the saikosaponin components in the bupleurum tenue particles as claimed in claim 1, wherein in S2, the chromatographic column Thermo Hypersil GOLD has a column length of 15cm, an inner diameter of 4.6mm, and a filler particle size of 3 μm.
5. The method for detecting the saikosaponin components in the bupleurum tenue particles as claimed in claim 1, wherein octadecylsilane chemically bonded silica is used as a filler in the chromatographic column Waters CORTECTS C18 or the chromatographic column Thermo Hypersil GOLD.
6. The method for detecting the saikosaponin components in the bupleurum tenue particles as claimed in claim 1, wherein when the mobile phase A adopts an aqueous solution containing 0.01% of acetic acid or 0.01% of formic acid, and the mobile phase B adopts chromatographic pure acetonitrile, the elution gradient is as follows:
0～55min，30～32％B；
55～58min，32～90％B；
58～61min，90～30％B；
61～70min，30％B。
7. the method for detecting the saikosaponin component in the bupleurum tenue particles according to claim 1, wherein when the mobile phase A adopts a 10% acetonitrile aqueous solution containing 0.1mmol of ammonium acetate (pH 4.0) and the mobile phase B adopts a 90% acetonitrile aqueous solution containing 0.1mmol of ammonium acetate (pH 4.0), the elution gradient is as follows:
0～35min，25～35％B；
35～37min，35～90％B；
37～45min，90～25％B；
45～50min，25％B。
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| CN113138239A (en) * | 2021-04-19 | 2021-07-20 | 华润三九医药股份有限公司 | Small radix bupleuri compound preparation fingerprint spectrum and construction method thereof and content determination method of small radix bupleuri compound preparation |
| CN113281449A (en) * | 2021-04-21 | 2021-08-20 | 广东志道医药科技有限公司 | Thin-layer identification method of bupleurum tenue preparation |
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