Abstract: A method for the analysis of trace taxanes in Taxus chinensis extracts by reversed-phase high performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry was developed. By studying the retention time and mass spectrometric characteristics of 10 known taxanes, a database of taxanes was established, and these data were used to quickly identify known taxanes in Taxus extract. And can do a certain degree of structural analysis of unknown compounds. This research method also has a guiding role in the analysis of other natural products, especially trace components.
Key words: paclitaxel, atmospheric pressure chemical ionization mass spectrometry, taxanes. Introduction Paclitaxel is a diterpenoid isolated from the stem bark of Taxus brevifolia, which is used to treat stubborn ovarian cancer. Breast cancer. However, paclitaxel resources are limited, which greatly limits its clinical application. Among the taxanes, many have antitumor activity, and some compounds that can be structurally engineered to synthesize paclitaxel are even more potent. Whether it is the study of taxanes in Taxus extracts or intermediates in structural engineering, efficient and sensitive separation and identification methods are needed. High performance liquid chromatography was used earlier for the separation of taxanes and the quantitative analysis of paclitaxel. Mass spectrometry has also been widely used in the analysis and identification of taxanes. The cation positive pressure atmospheric pressure chemical ionization mass spectrometry of taxanes was studied. The mass spectrometric characteristics of these materials were found. The quantitative analysis of the extracts of Taxus chinensis by reversed-phase high performance liquid chromatography and atmospheric pressure chemical ionization mass spectrometry was established. A method of a taxane compound.
2 Experimental 2.1 Reagents and instruments LCQ Advantage mass spectrometer (Thermo Finnigan, USA). Acetonitrile is HPLC pure; water is prepared by Milli-Q water purification system of Millipore Company of France; methanol, chloroform, n-hexane and ethyl acetate are analytically pure; silica gel for column chromatography (Shanghai Wusi Chemical Reagent Co., Ltd., 75-150 μm ). Bacaster III, 10-deacetylbaccatin III, paclitaxel, 10-deacetyl. 7 table paclitaxel, cephalosporin, 10-deacetyl. 7-Table of cephalosporin, 10-deacetyl-7-xylosyl cephalosporin, 10-deacetyl-7-xylosyl taxol, 10-deacetyl-7-xylosyl taxol C 7-epitaxol was prepared by our laboratory and confirmed by nuclear magnetic resonance. Mass spectrometry conditions: full range scanning 100-1000; shell gas (N2): 65 units, auxiliary gas (N2): 20 units; discharge current 5 μA; evaporator temperature 465 ° C, capillary temperature 180 ° C. Chromatographic conditions: MetaChem's Taxsil-3 column (250 mm x 4.6 mm); mobile phase: acetonitrile: water (49:51), flow rate: 0.8 mL/min, column temperature: 35 °C.
2.2 Sample treatment Take 1kg of dried Taxus mairei bark, pulverize to less than 250μm, extract with methanol 3 times (5L × 3), each time 10h, concentrate the extract, extract with n-hexane Three times (2 L × 3), the n-hexane layer was discarded, and then extracted three times with chloroform and water (2 L × 3), and the chloroform layer was concentrated and dried. On the silica gel column. The mobile phase was washed with ethyl acetate: n-hexane (1:1), and the corresponding fractions were collected and evaporated.
3 Results and discussion 3.1 Analysis of known taxanes By analyzing existing taxanes, a database of taxanes can be initially established, so that the yew extract can be analyzed quickly and conveniently. ,mid product. The molecular structure information, molecular weight and relative retention time of 10 common taxanes are listed. These taxanes were analyzed by liquid chromatography under the above conditions to obtain relative retention times of the respective compounds, that is, chromatographic characteristic data of these compounds, thereby facilitating comparison of data between the laboratories. The length of retention of the compound in reversed-phase chromatography can also be roughly measured as its lipophilic strength, which helps to explain the structure of the unknown taxane compound.
In the liquid chromatography-mass spectrometry, the molecular weight of the related compound and the fragment ions related to the structure can be obtained by mass spectrometry. Combined with the information on the lipophilicity of the compound obtained by liquid phase analysis, the structure can be quickly identified. The ion fragment ions of the 10 compounds illustrate how these fragment ions are produced. The mass spectra of these compounds have many commonalities. All of these compounds have the same parent ring structure as paclitaxel. They are formed after zoi, and the m/z=509 parent ring characteristic ions; each compound is also generated due to the different substituents on the parent ring. Many characteristic ions mainly include the following three types: *The class is about the characteristic ion of the 13-position upper side chain, and the side chain of many compounds is similar to paclitaxel (m/z=286), and only the functional group attached to the amino group (R1) ) There is a difference. This can be judged based on the charge-to-mass ratio of the side chain ions, such as 1O-deacetyl. 7-xyxy-based paclitaxel C replaces the phenyl group of paclitaxel with pentylene, so its characteristic ion of side chain is m/z=280, which is 6 smaller than paclitaxel; secondly, the side chain of cephalosporin, amino group Attached to a 2-butenyl group, so m / z = 264; there are some groups in the 13 position without a macromolecule, there will be no corresponding characteristic ions, such as 10-acetyl bakatan 11I and Pakistan Kating III, they are all hydroxyl at position 13, so there is no corresponding fragment ion. The second type is about the group attached to the 10 position. Since the group at the 1O position can form a stable energy structure with a lower energy bond with the double bond at the 11 position, the group at the 10 position is easily removed. To form a corresponding neutral fragment loss, if the 10-position is an acetyl group, the excimer ion can easily form a 60-neutral fragment loss. If the hydroxyl group is connected, the neutral fragment of 18 is easily lost. The third type is about the 7-position. Many compounds have the same functional group at the 7-position, except that the spatial positions of the bonds are different. The mass spectra of these compounds are basically the same. Considering that most taxanes have a similar structure to paclitaxel, especially the parent ring structure of paclitaxel, these substances undergo similar cleavage pathways to paclitaxel and other standard taxanes. The same characteristic ions and neutral molecules are lost, indicating that they contain the same structure, and the corresponding different ions indicate structural differences. Therefore, fine structure analysis of such compounds can be performed using APCI/MS.
3.2 Liquid-mass spectrometry analysis of Taxus extract 3.2.1 Determination of molecular weight of unknown compounds Figure 2 is a UV image (227 nm) and total mass spectrometry of an intermediate product in the purification process of paclitaxel. The two images are highly consistent, and most of the compounds are well separated by high performance liquid chromatography, creating good conditions for mass spectrometry. Due to the complexity of the composition of the product, and the inability of some compounds to achieve complete separation, the mass spectrometry capability of the mass spectrometry can be used for analysis. Although there is interference with other compound ion peaks, it can be judged that the retention time is 14.18 min, which is cephalosporin.
3.2.2 Preliminary analysis of taxanes in extracts To date, more than 300 taxane compounds have been discovered. The molecular weight of these compounds can be known from the structural formula. The selective ion scan of the mass spectrometer can be used to determine whether a certain mixture contains these compounds, and then confirm according to the spectrum of the mass spectrum. A chromatogram of some of the possible taxanes found by analysis of the intermediates by selective ion scanning. For new compounds that have not yet been discovered, some judgments can be made on the structure based on the mass spectrum. For example, whether or not the compound has the same parent ring as paclitaxel can be judged based on whether or not the compound forms a parent ring characteristic ion of m/z = 509.
3.2.3 Mass Spectrometry Analysis of Unknown Compounds Because many taxanes have similar structures and similar cleavage pathways, APCI/MS can be used to perform fine structure analysis of such compounds. Taking the unknown compound with a peak time of 7.21 min as an example, a method for analyzing the structure of a compound by mass spectrometry is described. The figure shows the atmospheric pressure chemical ionization mass spectrum of the compound, the mass spectrum of paclitaxel in the comparison chart: its side chain forms an ion with m/z = 268.1, and its parent ring forms m/z 509.1 and 527.1 after the side chain is de-chained. The ion indicates that the side chain of the compound is the same as that of paclitaxel, while the parent ring contains a structure similar to l0-deacetyl-7 table-paclitaxel; the excimer ion peak has m/z 943.9, which is better than l0-deacetyl-paclitaxel. Large 132.1, and there is an ion peak of m/z=794.0 obtained by excimer ion peak in the spectrum of the material, and the peak time of the substance in the chromatogram (7.2l)
Min) earlier than paclitaxel (15.92min), indicating that the substance is weaker than paclitaxel; comprehensively these three items, based on the molecular weight information and the existing taxane compounds, can basically determine the ratio of the compound on the parent ring Paclitaxel has one more xylosyl group and may be l0-deacetyl-7-xylosyl-paclitaxel.
3.3 Conclusions In this experiment, liquid chromatography-atmospheric pressure chemical ionization mass spectrometry was used to study the standard samples of taxanes, and the liquid chromatography retention time, mass spectrometry characteristics and mass spectrometry of these compounds were obtained. A database of taxanes for the analysis of crude extracts of Taxus and intermediates in the purification of paclitaxel. The method used in this experiment is also applicable to the development and research of other natural products, especially for trace components. Analysis.
References
1 Wani M C, Taylor H L, Wal1 M E, Coggon P, McPhail A T. Am. Chem. Soc. , 1971, 93: 2325 ~ 2327
2 Rowinsky E k, Cazermve L A, Doneyeer R C. Nat. Cancer. Inst. , 1990, 82: 1247 ~ 1259.
3 Kingston D I, Samaranayake G, lvey C A. Nat. Prod. , 1990, 53: 1 ~ 12
4 Denis J N, Greene A E, Guenard D, Francoise G V, Mangatal L, Potier P. J. Am Chem. Soc. , 1988, 110 (17): 5917 ~ 5919
Key words: paclitaxel, atmospheric pressure chemical ionization mass spectrometry, taxanes. Introduction Paclitaxel is a diterpenoid isolated from the stem bark of Taxus brevifolia, which is used to treat stubborn ovarian cancer. Breast cancer. However, paclitaxel resources are limited, which greatly limits its clinical application. Among the taxanes, many have antitumor activity, and some compounds that can be structurally engineered to synthesize paclitaxel are even more potent. Whether it is the study of taxanes in Taxus extracts or intermediates in structural engineering, efficient and sensitive separation and identification methods are needed. High performance liquid chromatography was used earlier for the separation of taxanes and the quantitative analysis of paclitaxel. Mass spectrometry has also been widely used in the analysis and identification of taxanes. The cation positive pressure atmospheric pressure chemical ionization mass spectrometry of taxanes was studied. The mass spectrometric characteristics of these materials were found. The quantitative analysis of the extracts of Taxus chinensis by reversed-phase high performance liquid chromatography and atmospheric pressure chemical ionization mass spectrometry was established. A method of a taxane compound.
2 Experimental 2.1 Reagents and instruments LCQ Advantage mass spectrometer (Thermo Finnigan, USA). Acetonitrile is HPLC pure; water is prepared by Milli-Q water purification system of Millipore Company of France; methanol, chloroform, n-hexane and ethyl acetate are analytically pure; silica gel for column chromatography (Shanghai Wusi Chemical Reagent Co., Ltd., 75-150 μm ). Bacaster III, 10-deacetylbaccatin III, paclitaxel, 10-deacetyl. 7 table paclitaxel, cephalosporin, 10-deacetyl. 7-Table of cephalosporin, 10-deacetyl-7-xylosyl cephalosporin, 10-deacetyl-7-xylosyl taxol, 10-deacetyl-7-xylosyl taxol C 7-epitaxol was prepared by our laboratory and confirmed by nuclear magnetic resonance. Mass spectrometry conditions: full range scanning 100-1000; shell gas (N2): 65 units, auxiliary gas (N2): 20 units; discharge current 5 μA; evaporator temperature 465 ° C, capillary temperature 180 ° C. Chromatographic conditions: MetaChem's Taxsil-3 column (250 mm x 4.6 mm); mobile phase: acetonitrile: water (49:51), flow rate: 0.8 mL/min, column temperature: 35 °C.
2.2 Sample treatment Take 1kg of dried Taxus mairei bark, pulverize to less than 250μm, extract with methanol 3 times (5L × 3), each time 10h, concentrate the extract, extract with n-hexane Three times (2 L × 3), the n-hexane layer was discarded, and then extracted three times with chloroform and water (2 L × 3), and the chloroform layer was concentrated and dried. On the silica gel column. The mobile phase was washed with ethyl acetate: n-hexane (1:1), and the corresponding fractions were collected and evaporated.
3 Results and discussion 3.1 Analysis of known taxanes By analyzing existing taxanes, a database of taxanes can be initially established, so that the yew extract can be analyzed quickly and conveniently. ,mid product. The molecular structure information, molecular weight and relative retention time of 10 common taxanes are listed. These taxanes were analyzed by liquid chromatography under the above conditions to obtain relative retention times of the respective compounds, that is, chromatographic characteristic data of these compounds, thereby facilitating comparison of data between the laboratories. The length of retention of the compound in reversed-phase chromatography can also be roughly measured as its lipophilic strength, which helps to explain the structure of the unknown taxane compound.
In the liquid chromatography-mass spectrometry, the molecular weight of the related compound and the fragment ions related to the structure can be obtained by mass spectrometry. Combined with the information on the lipophilicity of the compound obtained by liquid phase analysis, the structure can be quickly identified. The ion fragment ions of the 10 compounds illustrate how these fragment ions are produced. The mass spectra of these compounds have many commonalities. All of these compounds have the same parent ring structure as paclitaxel. They are formed after zoi, and the m/z=509 parent ring characteristic ions; each compound is also generated due to the different substituents on the parent ring. Many characteristic ions mainly include the following three types: *The class is about the characteristic ion of the 13-position upper side chain, and the side chain of many compounds is similar to paclitaxel (m/z=286), and only the functional group attached to the amino group (R1) ) There is a difference. This can be judged based on the charge-to-mass ratio of the side chain ions, such as 1O-deacetyl. 7-xyxy-based paclitaxel C replaces the phenyl group of paclitaxel with pentylene, so its characteristic ion of side chain is m/z=280, which is 6 smaller than paclitaxel; secondly, the side chain of cephalosporin, amino group Attached to a 2-butenyl group, so m / z = 264; there are some groups in the 13 position without a macromolecule, there will be no corresponding characteristic ions, such as 10-acetyl bakatan 11I and Pakistan Kating III, they are all hydroxyl at position 13, so there is no corresponding fragment ion. The second type is about the group attached to the 10 position. Since the group at the 1O position can form a stable energy structure with a lower energy bond with the double bond at the 11 position, the group at the 10 position is easily removed. To form a corresponding neutral fragment loss, if the 10-position is an acetyl group, the excimer ion can easily form a 60-neutral fragment loss. If the hydroxyl group is connected, the neutral fragment of 18 is easily lost. The third type is about the 7-position. Many compounds have the same functional group at the 7-position, except that the spatial positions of the bonds are different. The mass spectra of these compounds are basically the same. Considering that most taxanes have a similar structure to paclitaxel, especially the parent ring structure of paclitaxel, these substances undergo similar cleavage pathways to paclitaxel and other standard taxanes. The same characteristic ions and neutral molecules are lost, indicating that they contain the same structure, and the corresponding different ions indicate structural differences. Therefore, fine structure analysis of such compounds can be performed using APCI/MS.
3.2 Liquid-mass spectrometry analysis of Taxus extract 3.2.1 Determination of molecular weight of unknown compounds Figure 2 is a UV image (227 nm) and total mass spectrometry of an intermediate product in the purification process of paclitaxel. The two images are highly consistent, and most of the compounds are well separated by high performance liquid chromatography, creating good conditions for mass spectrometry. Due to the complexity of the composition of the product, and the inability of some compounds to achieve complete separation, the mass spectrometry capability of the mass spectrometry can be used for analysis. Although there is interference with other compound ion peaks, it can be judged that the retention time is 14.18 min, which is cephalosporin.
3.2.2 Preliminary analysis of taxanes in extracts To date, more than 300 taxane compounds have been discovered. The molecular weight of these compounds can be known from the structural formula. The selective ion scan of the mass spectrometer can be used to determine whether a certain mixture contains these compounds, and then confirm according to the spectrum of the mass spectrum. A chromatogram of some of the possible taxanes found by analysis of the intermediates by selective ion scanning. For new compounds that have not yet been discovered, some judgments can be made on the structure based on the mass spectrum. For example, whether or not the compound has the same parent ring as paclitaxel can be judged based on whether or not the compound forms a parent ring characteristic ion of m/z = 509.
3.2.3 Mass Spectrometry Analysis of Unknown Compounds Because many taxanes have similar structures and similar cleavage pathways, APCI/MS can be used to perform fine structure analysis of such compounds. Taking the unknown compound with a peak time of 7.21 min as an example, a method for analyzing the structure of a compound by mass spectrometry is described. The figure shows the atmospheric pressure chemical ionization mass spectrum of the compound, the mass spectrum of paclitaxel in the comparison chart: its side chain forms an ion with m/z = 268.1, and its parent ring forms m/z 509.1 and 527.1 after the side chain is de-chained. The ion indicates that the side chain of the compound is the same as that of paclitaxel, while the parent ring contains a structure similar to l0-deacetyl-7 table-paclitaxel; the excimer ion peak has m/z 943.9, which is better than l0-deacetyl-paclitaxel. Large 132.1, and there is an ion peak of m/z=794.0 obtained by excimer ion peak in the spectrum of the material, and the peak time of the substance in the chromatogram (7.2l)
Min) earlier than paclitaxel (15.92min), indicating that the substance is weaker than paclitaxel; comprehensively these three items, based on the molecular weight information and the existing taxane compounds, can basically determine the ratio of the compound on the parent ring Paclitaxel has one more xylosyl group and may be l0-deacetyl-7-xylosyl-paclitaxel.
3.3 Conclusions In this experiment, liquid chromatography-atmospheric pressure chemical ionization mass spectrometry was used to study the standard samples of taxanes, and the liquid chromatography retention time, mass spectrometry characteristics and mass spectrometry of these compounds were obtained. A database of taxanes for the analysis of crude extracts of Taxus and intermediates in the purification of paclitaxel. The method used in this experiment is also applicable to the development and research of other natural products, especially for trace components. Analysis.
References
1 Wani M C, Taylor H L, Wal1 M E, Coggon P, McPhail A T. Am. Chem. Soc. , 1971, 93: 2325 ~ 2327
2 Rowinsky E k, Cazermve L A, Doneyeer R C. Nat. Cancer. Inst. , 1990, 82: 1247 ~ 1259.
3 Kingston D I, Samaranayake G, lvey C A. Nat. Prod. , 1990, 53: 1 ~ 12
4 Denis J N, Greene A E, Guenard D, Francoise G V, Mangatal L, Potier P. J. Am Chem. Soc. , 1988, 110 (17): 5917 ~ 5919
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