CN113588846B - HPLC method for simultaneously detecting contents of four components in Taxus media - Google Patents
HPLC method for simultaneously detecting contents of four components in Taxus media Download PDFInfo
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- 241001674343 Taxus x media Species 0.000 title claims abstract description 45
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 34
- OVMSOCFBDVBLFW-VHLOTGQHSA-N 5beta,20-epoxy-1,7beta,13alpha-trihydroxy-9-oxotax-11-ene-2alpha,4alpha,10beta-triyl 4,10-diacetate 2-benzoate Chemical compound O([C@@H]1[C@@]2(C[C@H](O)C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)O)C(=O)C1=CC=CC=C1 OVMSOCFBDVBLFW-VHLOTGQHSA-N 0.000 claims abstract description 73
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- 238000000926 separation method Methods 0.000 claims description 12
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- YWLXLRUDGLRYDR-ZHPRIASZSA-N 5beta,20-epoxy-1,7beta,10beta,13alpha-tetrahydroxy-9-oxotax-11-ene-2alpha,4alpha-diyl 4-acetate 2-benzoate Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](O)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 YWLXLRUDGLRYDR-ZHPRIASZSA-N 0.000 description 1
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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Abstract
The invention discloses an HPLC method for simultaneously detecting the contents of four components in Taxus media, and belongs to the technical field of detection. The invention discloses an HPLC method for simultaneously detecting the contents of four components in Taxus media, which comprises the steps of Taxus media sample pretreatment, test solution preparation and HPLC method detection of the contents of 10-DAB, baccatin III, 10-DAP and paclitaxel in a test sample. The invention can realize the synchronous detection of the contents of the four components of 10-DAB, baccatin III, 10-DAP and taxol in the taxus media only by using an HPLC method, has less consumption sample and reagent amount, saves energy, has high efficiency, and has the characteristics of accuracy and high reliability.
Description
Technical Field
The invention relates to the technical field of detection, in particular to an HPLC (high performance liquid chromatography) method for simultaneously detecting the contents of four components in Taxus media.
Background
Paclitaxel is a complex tetracyclic diterpenoid compound separated from taxus chinensis, is one of the most effective antitumor drugs after cisplatin and adriamycin, and has remarkable effects in the aspects of treating breast cancer, lung cancer, ovarian cancer and the like. The Taxus media is a natural hybrid of Taxus cuspidata and Taxus baccata, and is one of the main raw material trees recognized in the world for extracting paclitaxel. The paclitaxel is obtained by a chemical semisynthesis way, namely extracting a precursor substance of the paclitaxel from branches and leaves (renewable parts) of the taxus media, and then by a chemical synthesis method, has the characteristics of high purity and low cost, and is also one of the main ways for industrially producing the paclitaxel at present. In particular, the semi-synthesis method using 10-DAP as a precursor has the characteristics of short synthetic route, high synthetic rate, simple and convenient operation and the like.
At present, the analysis and content determination of 10-DAB, baccatin III, 10-DAP and paclitaxel in Taxus x media are mainly to establish a HPLC method for separation and analysis respectively, and a UPLC, HPLC-MS method and the like based on the separation and analysis. These methods are complicated, high in workload, and high in chemical reagent consumption and cost.
Therefore, it is an urgent problem to be solved by those skilled in the art to provide an HPLC method for simultaneously detecting the contents of four components in taxus media.
Disclosure of Invention
In view of the above, the invention provides an HPLC method for simultaneously detecting the contents of four components (10-DAB, baccatin III, 10-DAP and paclitaxel) in Taxus media.
In order to achieve the purpose, the invention adopts the following technical scheme:
an HPLC method for simultaneously detecting the contents of four components in Taxus media comprises the following steps:
(1) pretreatment of a Taxus media sample and preparation of a test solution:
drying Taxus media tissue at 40-60 deg.C to constant weight, and pulverizing into powder; accurately weighing Taxus media dry powder, adding 90% methanol according to a material-liquid ratio of 1:10-1:15(g/mL), ultrasonically extracting at 40-60 deg.C for 40-60min, centrifuging the soaking solution at 8000-;
(2) detecting the content of 10-DAB, baccatin III, 10-DAP and paclitaxel in the sample by HPLC;
conditions for HPLC gradient elution separation of 10-DAB, baccatin III, 10-DAP and paclitaxel were: adopts ZORBAXeclipse XDB-C18(4.6 mm. times.250 mm. times.5. mu.l) chromatography column with mobile phase ATwo reagents, alcohol and water, methanol: the water gradient elution procedure was: 0-2min, 35: 65; 2-6min, 55: 45, a first step of; 6-24min, 65: 35; 24-40min, 100: 0; 40-46min, 35: 65; the detection wavelength is 227nm, the column temperature is 40 ℃, the flow rate is 1ml/min, and the sample injection amount is 10 mul.
According to the detection conditions, the peaks of the four components of 10-DAB, baccatin III, 10-DAP and paclitaxel in the test solution can be simultaneously separated on the base line of the chromatogram.
According to the technical scheme, compared with the prior art, the HPLC method for simultaneously detecting the contents of the four components in the taxus media has the following beneficial effects:
(1) the method can synchronously detect and analyze four sample components of 10-DAB, baccatin III, 10-DAP and paclitaxel in Taxus x media by HPLC without using a mass spectrometer and an ultrahigh-phase liquid chromatogram, greatly improves the detection efficiency of taxane compounds, and saves time cost and economic cost for research, development and production of enterprises and scientific research institutions.
(2) The method adopts an improved sample pretreatment mode and a gradient elution method, and only two reagents of methanol and water are used in the whole detection process, so that the 10-DAB, the baccatin III, the 10-DAP and the taxol can be well separated on a base line on an HPLC chromatogram, and the method can simultaneously determine the contents of the 10-DAB, the baccatin III, the 10-DAP and the taxol in the taxus chinensis, reduce the reagent consumption and has strong reliability.
(3) The method of the invention controls the dosage of the sample and the reagent according to the feed-liquid ratio, can complete the determination of 10-DAB, baccatin III, 10-DAP and paclitaxel in Taxus media plant under the micro condition, reduces the consumption of the sample, and has the characteristics of saving and high efficiency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a graph showing the separation profiles of 10-DAB, baccatin III, 10-DAP and paclitaxel standard samples under gradient elution conditions according to the present invention;
FIG. 2 is a graph showing regression curves and equations for the 10-DAB standard of the present invention;
FIG. 3 is a graph showing regression curves and equations for the baccatin III standard of the present invention;
FIG. 4 is a graph showing regression curves and equations for the 10-DAP standard of the present invention;
FIG. 5 is a graph showing regression curves and equations for paclitaxel standards according to the present invention;
FIG. 6 is an HPLC chromatogram of a Taxus media sample in example 1 of the present invention;
FIG. 7 is an HPLC chromatogram of a Taxus media sample in example 2 of the present invention;
FIG. 8 is an HPLC chromatogram of a Taxus media sample in example 3 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The experimental material is Taxus media planted and cultivated in experimental nursery garden of plant institute of Chinese academy of sciences (Nanjing Zhongshan botanical garden) of Jiangsu province, and the method, instrument and reagent used in the experiment are conventional methods, instruments and reagents in the technical field.
The main experimental apparatus: an Agilent1100 model high performance liquid chromatograph (Agilent company, usa), a KQ5200DE model digital control ultrasonic cleaner (ultrasound instruments ltd, kunshan), a nitrogen blower (youning instruments ltd, hangzhou), an electrothermal blowing dry box (shanghai-heng-science instruments ltd), and a 3K15 model high-speed centrifuge (Sigma company, germany).
The reagents used were: the pretreatment reagents are respectively methanol (analytically pure) and ultrapure water, and the HPLC chromatographic reagents are respectively methanol (chromatographically pure) and ultrapure water; 10-DAB standard (purity not less than 98%, CAS number: 32981-86-5), baccatin III standard (purity not less than 98%, CAS number: 27548-93-2), 10-DAP standard (purity not less than 98%, CAS number: 78432-77-6), purchased from Nanjing spring and autumn bioengineering, Inc.; paclitaxel standards (purity 99.9%, CAS number: 33069-62-4) were purchased from the Chinese food and drug testing institute.
Example 1
An HPLC method for simultaneously detecting the contents of four components in Taxus media comprises the following steps:
drying branches and leaves of Taxus media in an oven at 40 deg.C to constant weight, and grinding into powder; accurately weighing 0.1g of Taxus media dry powder, adding 1.5ml of 90% methanol, mixing, and ultrasonically extracting the mixed solution in an ultrasonic cleaner at 40 deg.C for 60 min; centrifuging the extracted soak solution at 10000r/min for 7min, collecting supernatant, pouring into a new centrifuge tube, volatilizing by a nitrogen blower, adding 1ml methanol for dissolving, and filtering the solution with 0.22 μm microporous membrane to obtain test solution.
Subjecting the test solution to high performance liquid chromatography with ZORBAXeclipse XDB-C18(4.6 mm. times.250 mm. times.5. mu.l) chromatography column with mobile phase of two reagents, methanol and water, methanol: the water gradient elution procedure was: 0-2min, 35: 65; 2-6min, 55: 45, a first step of; 6-24min, 65: 35; 24-40min, 100: 0; 40-46min, 35: 65; the detection wavelength is 227nm, the column temperature is 40 ℃, the flow rate is 1ml/min, and the sample injection amount is 10 mul.
Example 2
An HPLC method for simultaneously detecting the contents of four components in Taxus media comprises the following steps:
drying branches and leaves of Taxus media in an oven at 60 deg.C to constant weight, and grinding into powder; accurately weighing 0.2g of Taxus media dry powder, adding 2ml of 90% methanol, mixing, and ultrasonically extracting the mixture in an ultrasonic cleaner at 60 deg.C for 40 min; centrifuging the extracted soak solution at 8000r/min for 10min, collecting supernatant, pouring into a new centrifuge tube, volatilizing with nitrogen blower, dissolving in 1ml methanol, and filtering with 0.22 μm microporous membrane to obtain test solution.
Subjecting the test solution to high performance liquid chromatography with ZORBAX Eclipse XDB-C18(4.6 mm. times.250 mm. times.5. mu.l) chromatography column with mobile phase of two reagents, methanol and water, methanol: the water gradient elution procedure was: 0-2min, 35: 65; 2-6min, 55: 45, a first step of; 6-24min, 65: 35; 24-40min, 100: 0; 40-46min, 35: 65; the detection wavelength is 227nm, the column temperature is 40 ℃, the flow rate is 1ml/min, and the sample injection amount is 10 mul.
Example 3
An HPLC method for simultaneously detecting the contents of four components in Taxus media comprises the following steps:
drying branches and leaves of Taxus media in an oven at 50 deg.C to constant weight, and grinding into powder; accurately weighing 0.2g of Taxus media dry powder, adding 2.5ml of 90% methanol, mixing, and ultrasonically extracting the mixed solution in an ultrasonic cleaner at 50 deg.C for 50 min; centrifuging the extracted soak solution at 9000r/min for 8min, collecting supernatant, pouring into new centrifuge tube, volatilizing with nitrogen blower, dissolving in 1ml methanol, and filtering with 0.22 μm microporous membrane to obtain test solution.
Subjecting the test solution to high performance liquid chromatography with ZORBAX Eclipse XDB-C18(4.6 mm. times.250 mm. times.5. mu.l) chromatography column, mobile phase of two reagents methanol and water, methanol: the water gradient elution procedure was: 0-2min, 35: 65; 2-6min, 55: 45, a first step of; 6-24min, 65: 35; 24-40min, 100: 0; 40-46min, 35: 65; the detection wavelength is 227nm, the column temperature is 40 ℃, the flow rate is 1ml/min, and the sample injection amount is 10 mul.
Preparation and calculation of standard curves in examples 1 to 3: the purity of 10-DAB, baccatin III and 10-DAP standard substances is more than or equal to 98 percent, the purity of paclitaxel standard substances is 99.9 percent, 1.80mg of 10-DAB standard substances, 2.36mg of baccatin III standard substances, 2.18mg of 10-DAP standard substances and 4.26mg of paclitaxel standard substances are accurately weighed respectively, firstly, 10-DAB, baccatin III, 10-DAP and paclitaxel are dissolved by methanol and the volume is determined to 25ml, and the solution is used as standard substance mother liquor. Respectively injecting 2 mul, 5 mul, 10 mul, 15 mul and 30 mul of mother liquor of the standard substance solution according to the chromatographic conditions for detection and analysis, measuring the peak area of the mother liquor by liquid chromatography, respectively drawing a standard curve and a regression equation of 10-DAB, baccatin III, 10-DAP and paclitaxel by taking the peak area as a vertical coordinate (y) and the mass concentration of the 10-DAB, the baccatin III, the 10-DAP and the paclitaxel as a horizontal coordinate (x), and calculating the content and the concentration of the 10-DAB, the baccatin III, the 10-DAP and the paclitaxel in the sample by referring to the standard curve and the regression equation. In addition, according to the peak area integral conditions of the standard sample separation spectrum and the sample separation spectrum, the concentration of the standard mother solution is properly diluted, and the calculation range of the concentration of the standard curve is adjusted.
FIG. 1 is a separation spectrum of a mixed standard sample of 10-DAB, baccatin III, 10-DAP and paclitaxel under a gradient elution condition, and it can be known from the diagram that the peak emergence times of the 10-DAB, baccatin III, 10-DAP and paclitaxel standard sample are respectively 10.94min, 13.14min, 27.84min and 29.66min, and the 10-DAB, baccatin III, 10-DAP and paclitaxel are well separated from each other on an HPLC chromatogram without the existence of interference peaks.
Taking the mass concentrations of 10-DAB, baccatin III, 10-DAP and paclitaxel as abscissa x and the peak areas of 10-DAB, baccatin III, 10-DAP and paclitaxel as ordinate y, performing linear regression to obtain standard curves, regression equations and R of 10-DAB, baccatin III, 10-DAP and paclitaxel2. Wherein, FIGS. 2-5 show the standard curves, regression equations and R of the 10-DAB, baccatin III, 10-DAP and paclitaxel standards, respectively2. The linear regression equations obtained for 10-DAB, baccatin III, 10-DAP and paclitaxel were respectively, y-18.742 x-8.7437(R2＝0.9999)，y＝16.383x+0.5847(R2＝0.9983)，y＝23.591x-8.4552(R2＝0.9994)，y＝19.83x+40.046(R20.9979) indicating 10-DAB at 8.45-126.75 μ g/ml-1Has good linear relation in the range of 0.37-5.55 mug/ml for baccatin III-1Has good linear relation in the interior, 10-DAP is 3.42-51.27 mu g/ml-1Has good linear relation, paclitaxel is in17.02-102.14μg·ml-1With a good linear relationship within.
FIG. 6 is an HPLC chromatogram of a Taxus media sample of example 1. As can be seen from the figure, the peak emergence times of 10-DAB, baccatin III, 10-DAP and paclitaxel are respectively 10.92min, 13.29min, 27.25min and 29.16min, the chromatogram peak shapes are smooth and symmetrical, no interference peak exists, and good baseline separation is obtained. The contents of 10-DAB, baccatin III, 10-DAP and taxol in Taxus x media are 0.287mg g/g respectively by measurement and calculation with standard curve-1、0.010mg·g-1、0.147mg·g-1、0.278mg·g-1. The method has the advantages of high-efficiency separation and detection of the content of 4 taxane components in the Taxus media, high chromatogram reliability and greatly improved detection efficiency of taxane compounds.
FIG. 7 is an HPLC chromatogram of a Taxus media sample of example 2. As can be seen from the figure, the peak emergence times of 10-DAB, baccatin III, 10-DAP and paclitaxel are respectively 10.90min, 13.26min, 27.34min and 29.23min, the chromatogram peak shapes are smooth and symmetrical, no interference peak exists, and good baseline separation is obtained. The contents of 10-DAB, baccatin III, 10-DAP and taxol in Taxus x media were 0.296mg g/g respectively as measured by substituting into standard curve-1、0.014mg·g-1、0.155mg·g-1、0.288mg·g-1. According to the results of the embodiment 1 and the embodiment 2, the method effectively separates and detects the content of 4 taxane components in the taxus media, the chromatogram reliability is high, and the detection efficiency of the taxane compounds is greatly improved.
FIG. 8 is an HPLC chromatogram of a Taxus media sample of example 3. As can be seen from the figure, the peak emergence times of 10-DAB, baccatin III, 10-DAP and paclitaxel are respectively 10.88min, 13.25min, 27.38min and 29.27min, the chromatogram peak shapes are smooth and symmetrical, no interference peak exists, and good baseline separation is obtained. The contents of 10-DAB, baccatin III, 10-DAP and taxol in Taxus media are 0.299mg g-1、0.012mg·g-1、0.151mg·g-1、0.286mg·g-1. The results of FIGS. 6-8 showThe method of the embodiment 1-3 of the invention efficiently separates and detects the content of 4 taxane components in the taxus media, the chromatogram has high reliability, and the detection efficiency of the taxane compounds is greatly improved.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (1)
1. An HPLC method for simultaneously detecting the contents of four components in Taxus media is characterized by comprising the following specific steps:
(1) pretreatment of a Taxus media sample and preparation of a test solution:
drying Taxus media tissue at 40-60 deg.C to constant weight, and pulverizing into powder; accurately weighing Taxus media dry powder, adding 90% methanol according to a material-liquid ratio of 1:10-1:15 g/mL, performing ultrasonic extraction at 40-60 ℃ for 40-60min, centrifuging the soaking solution at 8000-;
(2) detecting the content of 10-DAB, baccatin III, 10-DAP and paclitaxel in the sample by HPLC;
conditions for HPLC gradient elution separation of 10-DAB, baccatin III, 10-DAP and paclitaxel were: ZORBAX Eclipse XDB-C is adopted184.6mm × 250mm × 5 μm, chromatography column, mobile phase of two reagents methanol and water, methanol: the water gradient elution procedure was: 0-2min, 35: 65; 2-6min, 55: 45, a first step of; 6-24min, 65: 35; 24-40min, 100: 0; 40-46min, 35: 65; the detection wavelength is 227nm, the column temperature is 40 ℃, the flow rate is 1ml/min, and the sample injection amount is 10 mul.
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