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WO2023019670A1 - 一种荧光素标记的核苷三磷酸的纯化方法 - Google Patents

一种荧光素标记的核苷三磷酸的纯化方法 Download PDF

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WO2023019670A1
WO2023019670A1 PCT/CN2021/118893 CN2021118893W WO2023019670A1 WO 2023019670 A1 WO2023019670 A1 WO 2023019670A1 CN 2021118893 W CN2021118893 W CN 2021118893W WO 2023019670 A1 WO2023019670 A1 WO 2023019670A1
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mobile phase
stage
component
fluorescein
composition
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PCT/CN2021/118893
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French (fr)
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何玲
蒋析文
兰诗龙
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广州达安基因股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • 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
    • 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
    • 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
    • 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
    • G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
    • 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

Definitions

  • the embodiment of the present invention relates to the field of biomedicine, in particular to a method for purifying fluorescein-labeled nucleoside triphosphates.
  • Fluorescence in situ hybridization is a non-radioactive in situ hybridization technology developed on the basis of the original radioactive in situ hybridization technology in the early 1980s. Bauman labeled RNA with fluorescence and used it as a probe for DNA detection.
  • the basic principle is to use a known labeled single-stranded nucleic acid as a probe, and according to the principle of base complementarity, heterosexually combine with the unknown single-stranded nucleic acid in the material to be tested to form a hybrid double-stranded nucleic acid that can be detected.
  • the probe can directly hybridize with the chromosome to locate a specific gene on the chromosome.
  • fluorescence in situ hybridization has the advantages of safety, rapidity, high sensitivity, strong detection signal, high hybridization specificity, and the ability to display multiple colors at the same time. Interphase nuclei are shown.
  • Fluorescence in situ hybridization mainly includes chromosome delineation, multi-color in situ initiation labeling, comparative genomic hybridization, fluorescence in situ hybridization on DNA fibers, etc., among which multi-color in situ initiation labeling is performed with oligonucleotides.
  • in situ PCR amplifies the sequence to be tested, and in the process, fluorescein directly or indirectly labeled nucleoside triphosphates are incorporated, so that the amplified sequence can be labeled.
  • the in situ amplification products of several sequences to be detected are labeled with different fluorescein, so as to realize the simultaneous detection of multiple small deletions and mutations and other minor changes in chromosomes.
  • This method has been routinely used for detection Localization of specific microsatellite sequences in chromosomes and interphase nuclei. It is more common for the incorporated nucleoside triphosphates to be directly labeled with fluorescein, which can ensure that each specific nucleotide incorporated is modified with fluorescein, and the stability of the signal is better.
  • the inventors have found that at least the following problems exist in the prior art: when preparing fluorescein-labeled nucleoside triphosphates, the crude product is purified and separated by reverse-phase high-performance liquid chromatography (HPLC), but the final product after separation is fluorescent. Poor intensity and insufficient sensitivity cannot meet the needs of fluorescence in situ hybridization experiments. Therefore, there is a need to develop a more efficient method for purifying crude fluorescein-labeled nucleoside triphosphates.
  • HPLC high-performance liquid chromatography
  • the purpose of the embodiments of the present invention is to provide a method for purifying fluorescein-labeled nucleoside triphosphates, so that the prepared fluorescein-labeled nucleoside triphosphates have higher purity.
  • an embodiment of the present invention provides a method for purifying fluorescein-labeled nucleoside triphosphates, comprising the following steps:
  • the mobile phase includes a first component and a second component, and the first component is n-hexylamine acetate aqueous solution, the second component is acetonitrile or methanol.
  • the crude product of fluorescein-labeled nucleoside triphosphate is obtained by reacting fluorescein and nucleoside triphosphate in a borate solution, and is purified by reversed-phase liquid chromatography.
  • the mobile phase in liquid chromatography is triethylamine acetate and acetonitrile.
  • the method for removing the solvent comprises the step of: performing rotary evaporation on the target fraction.
  • the mobile phase consists of the first component and the second component.
  • the first component is n-hexylamine acetate aqueous solution
  • the second component is acetonitrile
  • the volume ratio of the first component is not less than 50%; more preferably not less than 60%, more preferably not less than 65%, more preferably Preferably not less than 70%.
  • the volume ratio of the first component is no more than 70%, more preferably no more than 65%, more preferably no more than 60%, more preferably Preferably not more than 50%.
  • the volume of the first component accounts for no less than 50% and no more than 70%.
  • the liquid chromatography employs an isocratic elution procedure.
  • the liquid chromatography employs a gradient elution procedure.
  • the gradient elution program at least includes: an elution stage, the elution stage includes at least a first stage in which the composition of the mobile phase is constant and a second stage in which the composition of the mobile phase changes at a uniform rate;
  • the mobile phase has a first mobile phase composition
  • the mobile phase is composed of the first mobile phase and changes to the second mobile phase at a uniform speed
  • the time of the first stage is not less than 1 minute; more preferably not less than 2 minutes.
  • the time of the second stage is not less than 10 minutes, more preferably not less than 12 minutes; more preferably not less than 15 minutes; more preferably not less than 19 minutes; more preferably not less than 20 minutes minutes; more preferably not less than 23 minutes.
  • a third stage is also included after the second stage, and in the third stage, the mobile phase changes from the composition of the second mobile phase to the composition of the third mobile phase at a uniform speed, and the composition of the third mobile phase
  • a fourth stage is also included after the third stage, and in the fourth stage, the mobile phase is composed of the third mobile phase.
  • the time of the third stage is 0.05-0.15 minutes.
  • the duration of the fourth stage is 3-10 minutes, preferably 4-6 minutes.
  • an equilibrium stage is also included after the elution stage, and the equilibrium stage includes a fifth stage in which the composition of the mobile phase changes at a uniform speed and a sixth stage in which the composition of the mobile phase is constant;
  • the mobile phase is changed from the composition of the third mobile phase to the composition of the first mobile phase at a uniform speed
  • the mobile phase is composed of the first mobile phase.
  • the fifth stage lasts for 0.05-0.15 minutes.
  • the time of the sixth stage is 3-10 minutes, preferably 3-5 minutes.
  • the gradient elution program is as follows:
  • the gradient elution program is as follows:
  • the gradient elution procedure is as follows:
  • the concentration of the n-hexylamine acetate aqueous solution is 80-120 mM, such as 100 mM.
  • the flow rate of the mobile phase is 0.5-1.5 ml/min, more preferably 0.7-1.3 ml/min.
  • the chromatographic column is a C 18 chromatographic column, for example: Waters Xbridge BEH 4.6x250mm, 5um.
  • the column temperature of the chromatographic column is 24-26°C.
  • the embodiments of the present invention have at least the following advantages:
  • the method for purifying fluorescein-labeled nucleoside triphosphates has high efficiency for purifying fluorescein-labeled nucleoside triphosphates, and can combine the crude product of fluorescein-labeled nucleoside triphosphates with nucleosides Separation of nucleoside diphosphate target products with similar triphosphate structures that are difficult to separate, and obtaining fluorescein-labeled nucleoside triphosphates with a purity greater than 99%.
  • the fluorescein-labeled nucleoside triphosphate purified by the method for purifying fluorescein-labeled nucleoside triphosphate provided by the embodiment of the present invention has good fluorescence intensity and high sensitivity, and meets the needs of fluorescence in situ hybridization experiments.
  • Fig. 1 is the Fluorescein-12-dUTP product chromatogram prepared according to the method for the embodiment of the present invention 1;
  • Fig. 2 is the chromatogram of the refined product of Fluorescein-12-dUTP prepared according to the method of Example 2 of the present invention
  • Fig. 3 is the impurity mass spectrogram obtained in the method according to the embodiment of the present invention 2;
  • Fig. 4 is the mass spectrogram of Fluorescein-12-dUTP obtained in the method according to Example 2 of the present invention.
  • Fig. 5 is the chromatogram of the refined product of Fluorescein-12-dUTP prepared according to the method of Example 3 of the present invention.
  • Fig. 6 is the Tetramethyl-Rhodamine-5-dUTP product chromatogram prepared according to the method for embodiment 4 of the present invention.
  • Fig. 7 is the Tetramethyl-Rhodamine-5-dUTP refined product chromatogram prepared according to the method for Example 5 of the present invention.
  • Fig. 8 is the impurity mass spectrogram obtained in the method according to the embodiment of the present invention 5;
  • Fig. 9 is a mass spectrum of the Tetramethyl-Rhodamine-5-dUTP product obtained in the method according to Example 5 of the present invention.
  • the prepared fluorescein-labeled nucleoside triphosphate has poor fluorescence and luminescent effect and low sensitivity.
  • the inventor speculates that it may be due to insufficient purity of the fluorescein-labeled nucleoside triphosphate product, but the inventor failed to find impurities by using traditional liquid chromatography (the mobile phase is methanol-water system).
  • the inventors found that the mobile phase system was improved, using the first component of the present invention to be n-hexylamine acetate aqueous solution, and the second component to be a two-component mobile phase system of acetonitrile or methanol, the
  • the target fluorescein-labeled nucleoside triphosphate product is separated from impurities, and the inventor has determined the specific structure of each peak after separation by mass spectrometry (the impurity peak is fluorescein-labeled nucleoside diphosphate, and the target peak is fluorescein-labeled nucleoside triphosphoric acid), while the traditional traditional liquid chromatography (mobile phase is methanol-water system) cannot separate the impurity peak and the target peak.
  • the inventors developed a purification method for fluorescein-labeled nucleoside triphosphates, which is used to obtain products with better fluorescent luminescence effects and high sensitivity to meet the needs of in situ fluorescence hybridization experiments .
  • nucleoside triphosphate refers to a nucleotide containing three phosphate groups, including natural nucleoside triphosphates and deoxynucleoside triphosphates.
  • fluorescein refers to: well known to those skilled in the art, it refers to a chemical compound that emits light after being excited by light.
  • Non-limiting examples are CY5, EDANS, xanthine derivatives (e.g., fluorescein, rhodamine, Oregon green, HIV, Texas red), penem derivatives (e.g., indocyanine, oxazolam, Luo Huaqing), amber derivatives (eg, Seta, Se Tau, Square pigments), etc.
  • fluorescein-labeled nucleoside triphosphates refers to products obtained by directly or indirectly labeling nucleoside triphosphates with fluorescein, a non-limiting example being Fluorescein SFX-labeled nucleoside triphosphates.
  • liquid chromatography refers to a method of separating components in a mixture by utilizing their different affinities for the two phases of the stationary phase and the mobile phase.
  • high performance liquid chromatography is used in the present invention to separate the impurities in the crude product of fluorescein-labeled nucleoside triphosphate from the main product by utilizing the different affinity of each component to the stationary phase and the mobile phase.
  • gradient elution program refers to a method for separating and analyzing samples by continuously changing the concentration ratio of the mobile phase to a certain extent in the same analysis cycle.
  • isocratic elution procedure refers to a procedure in which a sample is separated and analyzed according to a constant mobile phase ratio in the same analysis cycle.
  • the term "constant change in the composition of the mobile phase” refers to a uniform change in the composition of the mobile phase over time, for example, the initial mobile phase composition is A component (90%)-B component (10%), in a Within minutes, the uniform speed changes to component A (80%)-component B (20%), that is, within one minute, component A decreases by 1.67% per second, while component B increases by 1.67% per second.
  • C 18 chromatographic column refers to a chromatographic column in which a reversed-phase column packing is based on silica gel and a non-polar octadecyl functional group structure is bonded on its surface.
  • High performance liquid chromatography passes a pressurized liquid solvent containing a sample mixture through a column packed with a solid adsorbent material, causing components of the sample to interact with the adsorbent material. As the different components interact differently with the adsorbent material, this results in a separation of the components as they exit the column.
  • a method for purifying fluorescein-labeled nucleoside triphosphates comprises the following steps:
  • the mobile phase includes a first component and a second component, and the first component is n-hexylamine acetate aqueous solution, the second component is acetonitrile or methanol.
  • the crude product of fluorescein-labeled nucleoside triphosphate is obtained by reacting fluorescein and nucleoside triphosphate in a borate solution, and is purified by reversed-phase liquid chromatography.
  • the mobile phase in liquid chromatography is triethylamine acetate and acetonitrile.
  • the method for removing the solvent comprises the step of: performing rotary evaporation on the target fraction.
  • the mobile phase consists of the first component and the second component.
  • the first component is n-hexylamine acetate aqueous solution
  • the second component is acetonitrile
  • the volume ratio of the first component is not less than 50%; more preferably not less than 60%, more preferably not less than 65%, more preferably Preferably not less than 70%.
  • the volume ratio of the first component is no more than 70%, more preferably no more than 65%, more preferably no more than 60%, more preferably Preferably not more than 50%.
  • the volume of the first component accounts for no less than 50% and no more than 70%.
  • the liquid chromatography employs an isocratic elution procedure.
  • the liquid chromatography employs a gradient elution procedure.
  • the gradient elution program at least includes: an elution stage, the elution stage includes at least a first stage in which the composition of the mobile phase is constant and a second stage in which the composition of the mobile phase changes at a uniform rate;
  • the mobile phase has a first mobile phase composition
  • the mobile phase is composed of the first mobile phase and changes to the second mobile phase at a uniform speed
  • the time of the first stage is not less than 1 minute; more preferably not less than 2 minutes.
  • the time of the second stage is not less than 10 minutes, more preferably not less than 12 minutes; more preferably not less than 15 minutes; more preferably not less than 19 minutes; more preferably not less than 20 minutes minutes; more preferably not less than 23 minutes.
  • a third stage is also included after the second stage, and in the third stage, the mobile phase changes from the composition of the second mobile phase to the composition of the third mobile phase at a uniform speed, and the composition of the third mobile phase
  • a fourth stage is also included after the third stage, and in the fourth stage, the mobile phase is composed of the third mobile phase.
  • the time of the third stage is 0.05-0.15 minutes.
  • the duration of the fourth stage is 3-10 minutes, preferably 4-6 minutes.
  • an equilibrium stage is also included after the elution stage, and the equilibrium stage includes a fifth stage in which the composition of the mobile phase changes at a uniform speed and a sixth stage in which the composition of the mobile phase is constant;
  • the mobile phase is changed from the composition of the third mobile phase to the composition of the first mobile phase at a uniform speed
  • the mobile phase is composed of the first mobile phase.
  • the fifth stage lasts for 0.05-0.15 minutes.
  • the time of the sixth stage is 3-10 minutes, preferably 3-5 minutes.
  • the gradient elution program is as follows:
  • the gradient elution program is as follows:
  • the gradient elution procedure is as follows:
  • the concentration of the n-hexylamine acetate aqueous solution is 80-120 mM, such as 100 mM.
  • the flow rate of the mobile phase is 0.5-1.5 ml/min, more preferably 0.7-1.3 ml/min.
  • the chromatographic column is a C 18 chromatographic column, for example: Waters Xbridge BEH 4.6x250mm, 5um.
  • the column temperature of the chromatographic column is 24-26°C.
  • the present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.
  • the conventional conditions or the conditions suggested by the manufacturer are usually followed. Percentages and parts are by weight unless otherwise indicated.
  • the experimental materials and reagents used in the following examples can be obtained from commercially available channels unless otherwise specified.
  • Embodiment 1 the preparation of Fluorescein-12-dUTP
  • reaction solution was filtered with a 0.22um organic filter membrane, and after filtration, it was purified by reverse phase C18 (chromatographic column Waters Xbridge BEH 4.6x250mm, 5um, mobile phase A phase: 100mM triethylamine acetate (TEAA), phase B : acetonitrile), inject three times, collect the target fraction and then concentrate to obtain the purified product of Fluorescein-12-dUTP.
  • reverse phase C18 chromatographic column Waters Xbridge BEH 4.6x250mm, 5um, mobile phase A phase: 100mM triethylamine acetate (TEAA), phase B : acetonitrile
  • Triethylamine acetate is a single peak in the purification spectrogram under mobile phase conditions, and no impurity peaks appear.
  • Embodiment 2 the purification of Fluorescein-12-dUTP
  • the resulting chromatogram is shown in Figure 2.
  • the retention time is 16.294min for the nucleoside diphosphate impurity labeled by Fluorescein NHS
  • the retention time is 18.927min for the nucleoside triphosphate product labeled by Fluorescein NHS. It can be seen that the separation between the product and the impurities is good.
  • nucleoside diphosphate impurity negative ion mode
  • mass spectrogram sees Fig. 3
  • nucleoside triphosphate product the nucleoside triphosphate product of the Fluorescein NHS label of 18.927min retention time
  • Negative ion mode the obtained mass spectrum is shown in Figure 4
  • the theoretical molecular weight of the nucleoside triphosphate labeled with Fluorescein NHS is 994.69.
  • the molecular ion peak is 993.59 in the negative ion mode, and the structure is correct.
  • the theoretical molecular weight of the nucleoside diphosphate impurity labeled by Fluorescein NHS is 914.69.
  • the molecular ion peak is 913.05 in negative ion mode, and the structure is correct.
  • Embodiment 3 the purification of Fluorescein-12-dUTP
  • the retention time of 9.501min is the nucleoside diphosphate impurity labeled by Fluorescein NHS
  • the retention time of 13.510min is the product of nucleoside triphosphate labeled by Fluorescein NHS.
  • the separation between the product and the impurity is good.
  • Embodiment 4 the preparation of Tetramethyl-Rhodamine-5-dUTP
  • reaction solution was filtered with a 0.22um organic filter membrane, and after filtration, it was purified by reverse phase C18 (chromatographic column Waters Xbridge BEH 4.6x250mm, 5um, mobile phase A phase: 100mM triethylamine acetate (TEAA), phase B : acetonitrile), sample injection three times, concentrate after collecting the purpose fraction, obtain Tetramethyl-Rhodamine-5-dUTP purified product.
  • reverse phase C18 chromatographic column Waters Xbridge BEH 4.6x250mm, 5um, mobile phase A phase: 100mM triethylamine acetate (TEAA), phase B : acetonitrile
  • Triethylamine acetate is a single peak in the purification spectrum under mobile phase conditions.
  • Embodiment 5 the refining of Tetramethyl-Rhodamine-5-dUTP
  • the resulting chromatogram is shown in Figure 7.
  • the retention time of 19.025min is the Rhodamine NHS-labeled nucleoside diphosphate impurity
  • the retention time of 21.884min is the Rhodamine NHS-labeled nucleoside triphosphate product. It can be seen that the separation between the product and the impurity is good.
  • the theoretical molecular weight of the nucleoside triphosphate labeled with Rhodamine NHS is 936.67.
  • the molecular ion peak is 933.99 in the negative ion mode, and the structure is correct.
  • the theoretical molecular weight of the nucleoside diphosphate labeled with Rhodamine NHS is 856.67.
  • the molecular ion peak in negative ion mode is 854, and the structure is correct.

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Abstract

一种荧光素标记的核苷三磷酸的纯化方法，具体为，使用液相色谱法纯化荧光素标记的核苷三磷酸粗产品，并收集目标馏分；其中，流动相包括第一组分和第二组分，所述第一组分为醋酸正己胺水溶液，所述第二组分选自乙腈或甲醇。此纯化方法可将荧光素标记的核苷三磷酸粗产品中与核苷三磷酸结构相近难以分离的核苷二磷酸与目标产物分离，获得纯度大于99％的荧光素标记的核苷三磷酸。纯化后的荧光素标记的核苷三磷酸，荧光强度好，灵敏度高，满足荧光原位杂交实验的需要。

Description

一种荧光素标记的核苷三磷酸的纯化方法
相关申请交叉引用
本专利申请要求于2021年08月19日提交的、申请号为202110953803.9、发明名称为“一种荧光素标记的核苷三磷酸的纯化方法”的中国专利申请的优先权，上述申请的全文以引用的方式并入本文中。
技术领域
本发明实施例涉及生物医药领域，特别涉及一种荧光素标记的核苷三磷酸的纯化方法。
背景技术
荧光原位杂交(FISH)是20世纪80年代初期在原有的放射性原位杂交技术的基础上发展起来的一种非放射性原位杂交技术，Bauman将RNA标记上荧光后作为探针进行DNA检测。其基本原理是用已知的标记单链核酸为探针，按照碱基互补的原则，与待检材料中未知的单链核酸进行异性结合，形成可被检测的杂交双链核酸。由于DNA分子在染色体上是沿着染色体纵轴呈线性排列，因而可以探针直接与染色体进行杂交从而将特定的基因定位在染色体上。与传统的放射性标记原位杂交相比，荧光原位杂交具有安全、快速、灵敏度高、检测信号强、杂交特异性高、能同时显示多种颜色等优点，不但能显示中期分裂相，还能显示间期核。
荧光原位杂交(FISH)在方法上主要有染色体描绘、多彩色原位启动标记、比较基因组杂交、在DNA纤维上的荧光原位杂交等，其中多彩色原位启动标记是用寡核苷酸作为引物，原位PCR扩增待测序列，并在此过程中掺入荧光素直接或间接标记的核苷三磷酸，使扩增出的序列都得以标记。通过几轮这样的扩增，使待检的几个序列的原位扩增产物标记上不同荧光素，实现同时对多个微小缺失和突变等染色体微小改变的检测，该方法已常规用于检测特异性微卫星序列在染色体和间期核内的定位。其中掺入的核苷三磷酸以荧光素直接标记的较为常见，这样可以保证被掺入的每个特定核苷酸都有荧光素修饰，信号的稳定行更优。
发明人发现现有技术中至少存在如下问题：在制备荧光素标记的核苷三磷酸时，对粗产物采用反相高效液相色谱法(HPLC)进行纯化分离，但分离后终产品，荧光发光强度较差、灵敏度不足，无法满足荧光原位杂交实验的需要。因此，需要开发一种更有效的纯化荧光素标记的核苷三磷酸粗产物的方法。
发明内容
本发明实施方式的目的在于提供一种荧光素标记的核苷三磷酸的纯化方法，使得制备的荧光素标记的核苷三磷酸纯度更高。
为解决上述技术问题，本发明的实施方式提供了一种荧光素标记的核苷三磷酸的纯化方法，包括以下步骤：
使用液相色谱法纯化荧光素标记的核苷三磷酸粗产品，并收集目标馏分，去除溶剂，即得；其中，流动相包括第一组分和第二组分，所述第一组分为醋酸正己胺水溶液，所述第二组分为乙腈或甲醇。
在一些优选的方案中，所述荧光素标记的核苷三磷酸粗产品由荧光素和核苷三磷酸在硼酸盐溶液中反应，并通过反相液相色谱法提纯后获得，所述反相液相色谱法中流动相为醋酸三乙胺和乙腈。
在一些优选的方案中，所述去除溶剂的方法包括步骤：对目标馏分进行旋蒸。
在一些优选的方案中，所述流动相由第一组分和第二组分组成。
在一些优选的方案中，所述第一组分为醋酸正己胺水溶液，所述第二组分为乙腈。
在一些优选的方案中，相对于所述流动相的总体积，所述第一组分的体积占比不少于50％；更优选不少于60％，更优选不少于65％，更优选不少于70％。
在一些优选的方案中，相对于所述流动相的总体积，所述第一组分的体积占比不多于70％，更优选不多于65％，更优选不多于60％，更优选不多于50％。
在一些优选的方案中，相对于所述流动相的总体积，所述第一组分的体积占比不少于50％且不多于70％。
在一些优选的方案中，所述液相色谱法采用等度洗脱程序。
在一些优选的方案中，所述等度洗脱程序中，所述流动相的组成如下：所述第一组分体积:所述第二组分的体积＝a’:b’，其中，a’为60～80，b’为20～40，且a’+b’＝100。
在一些优选的方案中，所述液相色谱法采用梯度洗脱程序。
在一些优选的方案中，所述梯度洗脱程序中至少包括：洗脱阶段，所述洗脱阶段至少包括流动相的组成恒定的第一阶段以及流动相的组成匀速变化的第二阶段；
所述第一阶段中，所述流动相具有第一流动相组成，所述第一流动相组成为：所述第一组分体积:所述第二组分的体积＝a 1:b 1；
所述第二阶段中，所述流动相由第一流动相组成匀速变化至第二流动相组成，所述第二 流动相组成为：所述第一组分体积:所述第二组分的体积＝a 2:b 2；
其中，a 1为75～85，b 1为15～25，a 2为55～65，b 2为35～45，且a 1+b 1＝100，a 2+b 2＝100。
在一些优选的方案中，所述第一阶段的时间不少于1分钟；更优选不少于2分钟。
在一些优选的方案中，所述第二阶段的时间不少于10分钟，更优选不少于12分钟；更优选不少于15分钟；更优选不少于19分钟；更优选不少于20分钟；更优选不少于23分钟。
在一些优选的方案中，所述第二阶段后还包括第三阶段，所述第三阶段中，所述流动相由第二流动相组成匀速变化至第三流动相组成，所述第三流动相组成为：所述第一组分体积:所述第二组分的体积＝a 3:b 3；其中，a 3为8～12，b 3为88～92，且a 3+b 3＝100；
所述第三阶段后还包括第四阶段，所述第四阶段中，所述流动相为第三流动相组成。
在一些优选的方案中，所述第三阶段的时间为0.05～0.15分钟。
在一些优选的方案中，所述第四阶段的时间为3～10分钟，优选为4～6分钟。
在一些优选的方案中，所述洗脱阶段后还包括平衡阶段，所述平衡阶段中包括流动相的组成匀速变化的第五阶段和流动相的组成恒定的第六阶段；
所述第五阶段中，所述流动相由第三流动相组成匀速变化至第一流动相组成；
所述第六阶段中，所述流动相为第一流动相组成。
在一些优选的方案中，所述第五阶段的时间为0.05～0.15分钟。
在一些优选的方案中，所述第六阶段的时间为3～10分钟，优选为3～5分钟。
在一些优选的方案中，当流速为不大于1.3ml/min且不小于0.7ml/min时，所述梯度洗脱程序如下：
时间(min) 第一组分(％) 第二组分(％)
0 80 20
2 80 20
25 60 40
25.1 10 90
30 10 90
30.1 80 20
35 80 20
在一些优选的方案中，当流速为1.5ml/min时，所述梯度洗脱程序如下：
时间(min) 第一组分(％) 第二组分(％)
0 80 20
1 80 20
16 60 40
16.1 10 90
19 10 90
19.1 80 20
22 80 20
在一些优选的方案中，当流速为0.5ml/min时，所述梯度洗脱程序如下：
时间(min) 第一组分(％) 第二组分(％)
0 80 20
3 80 20
40 60 40
40.1 10 90
50 10 90
50.1 80 20
60 80 20
在一些优选的方案中，所述醋酸正己胺水溶液的浓度为80～120mM，例如100mM。
在一些优选的方案中，所述流动相的流速为0.5～1.5ml/min，更优选为0.7～1.3ml/min。
在一些优选的方案中，所述色谱柱为C 18色谱柱，例如：Waters Xbridge BEH 4.6x250mm,5um。
在一些优选的方案中，所述色谱柱的柱温为24～26℃。
本发明实施方式相对于现有技术而言，至少具有下述优点：
(1)本发明的实施方式提供的荧光素标记的核苷三磷酸的纯化方法，纯化荧光素标记的核苷三磷酸效率高，可将荧光素标记的核苷三磷酸粗产品中与核苷三磷酸结构相近难以分离的核苷二磷酸目标产物分离，获得纯度大于99％的荧光素标记的核苷三磷酸。
(2)经本发明的实施方式提供的荧光素标记的核苷三磷酸的纯化方法纯化的荧光素标记的核苷三磷酸，荧光强度好，灵敏度高，满足荧光原位杂交实验的需要。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
一个或多个实施例通过与之对应的附图中的图片进行示例性说明，这些示例性说明并不构成对实施例的限定。
图1是根据本发明实施例1的方法制备的Fluorescein-12-dUTP产品色谱图；
图2是根据本发明实施例2的方法制备的Fluorescein-12-dUTP精制产品色谱图；
图3是根据本发明实施例2的方法中所得杂质质谱图；
图4是根据本发明实施例2的方法中所得Fluorescein-12-dUTP质谱图；
图5是根据本发明实施例3的方法制备的Fluorescein-12-dUTP精制产品色谱图；
图6是根据本发明实施例4的方法制备的Tetramethyl-Rhodamine-5-dUTP产品色谱图；
图7是根据本发明实施例5的方法制备的Tetramethyl-Rhodamine-5-dUTP精制产品色谱图；
图8是根据本发明实施例5的方法中所得杂质质谱图；
图9是根据本发明实施例5的方法中所得Tetramethyl-Rhodamine-5-dUTP产品质谱图。
具体实施方式
现有技术中，制备获得的荧光素标记的核苷三磷酸荧光发光效果差，灵敏度低。发明人推测可能是由于荧光素标记的核苷三磷酸产品纯度不足所致，但发明人使用传统液相色谱法(流动相为甲醇-水体系)，并未能发现杂质。本发明人经过进一步的研究，发现对流动相体系进行改进，使用本发明所述的第一组分为醋酸正己胺水溶液、第二组分为乙腈或甲醇的二组分流动相体系，可以将目标荧光素标记的核苷三磷酸产品与杂质进行分离，发明人通过质谱确定了分离后各峰的具体结构(杂质峰为荧光素标记的核苷二磷酸、目标峰为荧光素标记的核苷三磷酸)，而传统的传统液相色谱法(流动相为甲醇-水体系)无法将杂质峰和目标峰进行分离。
在上述研究发现的基础上，发明人开发了一种荧光素标记的核苷三磷酸的纯化方法，用于获得荧光发光效果更好的，灵敏度高的产品，以满足原位荧光杂交实验的需要。
术语
如本文所用，术语“核苷三磷酸”指的是一种含有三个磷酸基团的核苷酸，包括自然型核苷三磷酸以及脱氧核苷三磷酸。
如本文所用，术语“荧光素”指的是：为本领域技术人员所熟知，指的是光激发后再辐 射光的化学化合物。非限制性示例为CY5，EDANS，黄嘌呤衍生物(例如，荧光素，罗丹明，Oregon green，爱滋病毒，Texas red)，青霉烯衍生物(例如，吲哚菁，恶唑安定，美罗花青)，琥珀色衍生物(例如，Seta，Se Tau，Square色素)等。
如本文所用，术语“荧光素标记的核苷三磷酸”指的是使用荧光素对核苷三磷酸进行直接或间接标记所得的产品，非限制性示例为Fluorescein SFX标记的核苷三磷酸。
如本文所用，术语“液相色谱法”指的是利用混合物中各组分对固定相和流动相两相的亲和力不同，实现混合物中各组分分离的方法。例如本发明中使用高效液相色谱仪，利用各组分对固定相和流动相的亲和力不同，将荧光素标记的核苷三磷酸粗产品中各杂质与主产物分离。
如本文所用，术语“梯度洗脱程序”指的是在同一个分析周期中，按照一定程度不断改变流动相的浓度配比，对样品进行分离分析的手段。
如本文所用，术语“等度洗脱程序”指的是在同一个分析周期中，按照不变的流动相配比对样品进行分离分析的程序。
如本文所用，术语“流动相的组成匀速变化”指的是流动相组成随着时间均匀的改变，例如初始流动相组成为A组分(90％)-B组分(10％)，在一分钟内，匀速变化至A组分(80％)-B组分(20％)，即为一分钟内，A组分每秒钟减少1.67％，同时B组分每秒钟增加1.67％。
如本文所用，术语“C 18色谱柱”指的是反相柱填料以硅胶为基质，在其表面键合非极性十八烷基官能团结构的色谱柱。
高效液相色谱法(HPLC)将含有样品混合物的加压液体溶剂通过填充有固体吸附材料的柱，导致样品的组分与吸附材料相互作用。由于不同的组分与吸附材料的相互作用不同，这导致组分在流出塔时分离。本发明提供的一种荧光素标记的核苷三磷酸的纯化方法，包括以下步骤：
使用液相色谱法纯化荧光素标记的核苷三磷酸粗产品，并收集目标馏分，去除溶剂，即得；其中，流动相包括第一组分和第二组分，所述第一组分为醋酸正己胺水溶液，所述第二组分为乙腈或甲醇。
在一些优选的方案中，所述荧光素标记的核苷三磷酸粗产品由荧光素和核苷三磷酸在硼酸盐溶液中反应，并通过反相液相色谱法提纯后获得，所述反相液相色谱法中流动相为醋酸三乙胺和乙腈。
在一些优选的方案中，所述去除溶剂的方法包括步骤：对目标馏分进行旋蒸。
在一些优选的方案中，所述流动相由第一组分和第二组分组成。
在一些优选的方案中，所述第一组分为醋酸正己胺水溶液，所述第二组分为乙腈。
在一些优选的方案中，相对于所述流动相的总体积，所述第一组分的体积占比不少于50％；更优选不少于60％，更优选不少于65％，更优选不少于70％。
在一些优选的方案中，相对于所述流动相的总体积，所述第一组分的体积占比不多于70％，更优选不多于65％，更优选不多于60％，更优选不多于50％。
在一些优选的方案中，相对于所述流动相的总体积，所述第一组分的体积占比不少于50％且不多于70％。
在一些优选的方案中，所述液相色谱法采用等度洗脱程序。
在一些优选的方案中，所述等度洗脱程序中，所述流动相的组成如下：所述第一组分体积:所述第二组分的体积＝a’:b’，其中，a’为60～80，b’为20～40，且a’+b’＝100。
在一些优选的方案中，所述液相色谱法采用梯度洗脱程序。
在一些优选的方案中，所述梯度洗脱程序中至少包括：洗脱阶段，所述洗脱阶段至少包括流动相的组成恒定的第一阶段以及流动相的组成匀速变化的第二阶段；
所述第一阶段中，所述流动相具有第一流动相组成，所述第一流动相组成为：所述第一组分体积:所述第二组分的体积＝a 1:b 1；
所述第二阶段中，所述流动相由第一流动相组成匀速变化至第二流动相组成，所述第二流动相组成为：所述第一组分体积:所述第二组分的体积＝a 2:b 2；
其中，a 1为75～85，b 1为15～25，a 2为55～65，b 2为35～45，且a 1+b 1＝100，a 2+b 2＝100。
在一些优选的方案中，所述第一阶段的时间不少于1分钟；更优选不少于2分钟。
在一些优选的方案中，所述第二阶段的时间不少于10分钟，更优选不少于12分钟；更优选不少于15分钟；更优选不少于19分钟；更优选不少于20分钟；更优选不少于23分钟。
在一些优选的方案中，所述第二阶段后还包括第三阶段，所述第三阶段中，所述流动相由第二流动相组成匀速变化至第三流动相组成，所述第三流动相组成为：所述第一组分体积:所述第二组分的体积＝a 3:b 3；其中，a 3为8～12，b 3为88～92，且a 3+b 3＝100；
所述第三阶段后还包括第四阶段，所述第四阶段中，所述流动相为第三流动相组成。
在一些优选的方案中，所述第三阶段的时间为0.05～0.15分钟。
在一些优选的方案中，所述第四阶段的时间为3～10分钟，优选为4～6分钟。
在一些优选的方案中，所述洗脱阶段后还包括平衡阶段，所述平衡阶段中包括流动相的组成匀速变化的第五阶段和流动相的组成恒定的第六阶段；
所述第五阶段中，所述流动相由第三流动相组成匀速变化至第一流动相组成；
所述第六阶段中，所述流动相为第一流动相组成。
在一些优选的方案中，所述第五阶段的时间为0.05～0.15分钟。
在一些优选的方案中，所述第六阶段的时间为3～10分钟，优选为3～5分钟。
在一些优选的方案中，当流速为不大于1.3ml/min且不小于0.7ml/min时，所述梯度洗脱程序如下：
时间(min) 第一组分(％) 第二组分(％)
0 80 20
2 80 20
25 60 40
25.1 10 90
30 10 90
30.1 80 20
35 80 20
在一些优选的方案中，当流速为1.5ml/min时，所述梯度洗脱程序如下：
时间(min) 第一组分(％) 第二组分(％)
0 80 20
1 80 20
16 60 40
16.1 10 90
19 10 90
19.1 80 20
22 80 20
在一些优选的方案中，当流速为0.5ml/min时，所述梯度洗脱程序如下：
时间(min) 第一组分(％) 第二组分(％)
0 80 20
3 80 20
40 60 40
40.1 10 90
50 10 90
50.1 80 20
60 80 20
在一些优选的方案中，所述醋酸正己胺水溶液的浓度为80～120mM，例如100mM。
在一些优选的方案中，所述流动相的流速为0.5～1.5ml/min，更优选为0.7～1.3ml/min。
在一些优选的方案中，所述色谱柱为C 18色谱柱，例如：Waters Xbridge BEH 4.6x250mm,5um。
在一些优选的方案中，所述色谱柱的柱温为24～26℃。为使本发明实施例的目的、技术方案和优点更加清楚，下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
除非另有指明，本文所用的技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义，需要注意的是，本文所用的术语仅为了描述具体实施方式，而非意图限制本申请的示例性实施方式。
实施例1、Fluorescein-12-dUTP的的制备
将5mg Fluorescein SFX(ThermoFisher)溶于100ul DMSO溶液中，振瑶5min溶解；将AA-dUTP 1mg(biotium)用300ul 0.1M PH9.0的硼酸盐振瑶5min溶解，取Fluorescein SFX 35ul加入到上述AA-dUTP的硼酸盐溶液中，常温振瑶反应2小时，结束反应。对反应液进行粗提纯，具体步骤如下：
将上述反应液用0.22um的有机过滤膜过滤，过滤后采用反相C 18的进行纯化(色谱柱Waters Xbridge BEH 4.6x250mm,5um，流动相A相：100mM醋酸三乙胺(TEAA)，B相：乙腈)，进样三次，收集目的馏分后进行浓缩，得Fluorescein-12-dUTP纯化品。
上述Fluorescein-12-dUTP纯化条件如表1：
表1
Figure PCTCN2021118893-appb-000001
Figure PCTCN2021118893-appb-000002
所得色谱图见图1。
醋酸三乙胺(TEAA)为流动相条件下的纯化谱图为单一的峰，未出现杂质峰。
但是后续实验发现，经上述方法制得的产品，荧光发光强度较差、灵敏度不足，无法满足荧光原位杂交实验的需要。经过大量测试和数据分析，发明人怀疑是由于Fluorescein-12-dUTP纯化品中存在未知杂质导致的。
实施例2、Fluorescein-12-dUTP的精制
将实施例1Fluorescein-12-dUTP纯化品用500ul去离子水溶解，按下表2条件进行精制：
表2
Figure PCTCN2021118893-appb-000003
所得色谱图见图2。其中保留时间为16.294min为Fluorescein NHS标记的核苷二磷酸杂质，保留时间为18.927min为Fluorescein NHS标记的核苷三磷酸产物，可见产物与杂质的分 离度良好。
通过LC-MS分别检测保留时间为16.294min的Fluorescein NHS标记的核苷二磷酸杂质(负离子模式)(质谱图见图3)、和保留时间为18.927min的Fluorescein NHS标记的核苷三磷酸产物(负离子模式)(所得质谱图见图4)。
根据上述质谱图数据，Fluorescein NHS标记的核苷三磷酸理论分子量为994.69，图4中，负离子模式下分子离子峰为993.59，结构正确。
Fluorescein NHS标记的核苷二磷酸杂质理论分子量量为914.69，图3中，负离子模式下分子离子峰为913.05，结构正确。
收集保留时间为17.5-19.5min的馏分，旋蒸去除流动相，即得纯化后的Fluorescein NHS标记的核苷三磷酸。
实施例3、Fluorescein-12-dUTP的精制
将实施例1所得Fluorescein-12-dUTP纯化品用500ul去离子水溶解，按下表3条件进行纯化：
表3
Figure PCTCN2021118893-appb-000004
所得色谱图见图5。其中保留时间为9.501min为Fluorescein NHS标记的核苷二磷酸杂质，保留时间为13.510min为Fluorescein NHS标记的核苷三磷酸产物，产物与杂质的分离度良好。
收集保留时间为12.5-16.0min的馏分，旋蒸去除流动相，即得纯化后的Fluorescein NHS标记的核苷三磷酸。
实施例4、Tetramethyl-Rhodamine-5-dUTP的制备
将5mg Rhodamine NHS溶于100ul DMSO溶液中，振瑶5min溶解；将AA-dUTP 1mg(biotium)用300ul 0.1M pH为9.0的硼酸盐振瑶5min溶解，取Rhodamine NHS 35ul加入到上述AA-dUTP的硼酸盐溶液中，常温振瑶反应2小时，结束反应。对反应液进行粗提纯，具体步骤如下：
将上述反应液用0.22um的有机过滤膜过滤，过滤后采用反相C 18的进行纯化(色谱柱Waters Xbridge BEH 4.6x250mm,5um，流动相A相：100mM醋酸三乙胺(TEAA)，B相：乙腈)，进样三次，收集目的馏分后进行浓缩，得Tetramethyl-Rhodamine-5-dUTP纯化品。
上述Tetramethyl-Rhodamine-5-dUTP纯化条件如表4：
表4
Figure PCTCN2021118893-appb-000005
所得色谱图见图6。
醋酸三乙胺(TEAA)为流动相条件下的纯化谱图为单一的峰。
实施例5、Tetramethyl-Rhodamine-5-dUTP的精制
将实施例4所得Tetramethyl-Rhodamine-5-dUTP纯化品用500ul去离子水溶解，按下表5条件进行精制：
表5
Figure PCTCN2021118893-appb-000006
所得色谱图见图7。其中保留时间为19.025min为Rhodamine NHS标记的核苷二磷酸杂质，保留时间为21.884min为Rhodamine NHS标记的核苷三磷酸产物，可见产物与杂质的分离度良好。
通过LC-MS分别检测保留时间为19.025min的Rhodamine NHS标记的核苷二磷酸杂质(负离子模式)(质谱图见图8)、和保留时间为21.884min的Rhodamine NHS标记的核苷三磷酸产物(负离子模式)(所得质谱图见图9)。
根据上述质谱图数据，Rhodamine NHS标记的核苷三磷酸理论分子量为936.67，图9中，负离子模式下分子离子峰为933.99，结构正确。
Rhodamine NHS标记的核苷二磷酸理论分子量为856.67，图8中，负离子模式下的分子离子峰为854，结构正确。
收集保留时间为21.00～22.00min的馏分，旋蒸去除流动相，即得纯化后的Rhodamine NHS标记的核苷三磷酸。
本领域的普通技术人员可以理解，上述各实施方式是实现本发明的具体实施例，而在实际应用中，可以在形式上和细节上对其作各种改变，而不偏离本发明的精神和范围。

Claims (10)

  1. 一种荧光素标记的核苷三磷酸的纯化方法，其特征在于，包括以下步骤：
    使用液相色谱法纯化荧光素标记的核苷三磷酸粗产品，并收集目标馏分；其中，流动相包括第一组分和第二组分，所述第一组分为醋酸正己胺水溶液，所述第二组分为乙腈或甲醇。
  2. 根据权利要求1所述的纯化方法，其特征在于，所述荧光素标记的核苷三磷酸粗产品由荧光素和核苷三磷酸在硼酸盐溶液中反应，并通过反相液相色谱法提纯后获得，所述反相液相色谱法中流动相为醋酸三乙胺和乙腈。
  3. 根据权利要求1所述的纯化方法，其特征在于，所述第一组分为醋酸正己胺水溶液，所述第二组分为乙腈。
  4. 根据权利要求3所述的纯化方法，其特征在于，所述液相色谱法采用等度洗脱程序；所述等度洗脱程序中，所述流动相的组成如下：所述第一组分体积:所述第二组分的体积＝a’:b’，其中，a’为60～80，b’为20～40，且a’+b’＝100。
  5. 根据权利要求3所述的纯化方法，其特征在于，所述液相色谱法采用梯度洗脱程序，所述梯度洗脱程序中至少包括：洗脱阶段，所述洗脱阶段至少包括流动相的组成恒定的第一阶段以及流动相的组成匀速变化的第二阶段；
    所述第一阶段中，所述流动相具有第一流动相组成，所述第一流动相组成为：所述第一组分体积:所述第二组分的体积＝a 1:b 1；
    所述第二阶段中，所述流动相由第一流动相组成匀速变化至第二流动相组成，所述第二流动相组成为：所述第一组分体积:所述第二组分的体积＝a 2:b 2；
    其中，a 1为75～85，b 1为15～25，a 2为55～65，b 2为35～45，且a 1+b 1＝100，a 2+b 2＝100。
  6. 根据权利要求5所述的纯化方法，其特征在于，所述第一阶段的时间不少于1分钟；
    和/或，所述第二阶段的时间不少于10分钟。
  7. 根据权利要求5所述的纯化方法，其特征在于，所述第二阶段后还包括第三阶段，所述第三阶段中，所述流动相由第二流动相组成匀速变化至第三流动相组成，所述第三流动相组成为：所述第一组分体积:所述第二组分的体积＝a 3:b 3；其中，a 3为8～12，b 3为88～92，且a 3+b 3＝100；
    所述第三阶段后还包括第四阶段，所述第四阶段中，所述流动相为第三流动相组成。
  8. 根据权利要求7所述的纯化方法，其特征在于，所述第三阶段的时间为0.05～0.15分钟；
    所述第四阶段的时间为3～10分钟。
  9. 根据权利要求6所述的纯化方法，其特征在于，所述洗脱阶段后还包括平衡阶段，所述平衡阶段中包括流动相的组成匀速变化的第五阶段和流动相的组成恒定的第六阶段；
    所述第五阶段中，所述流动相由第三流动相组成匀速变化至第一流动相组成；
    所述第六阶段中，所述流动相为第一流动相组成。
  10. 根据权利要求9所述的纯化方法，其特征在于，醋酸正己胺水溶液的浓度为80～120mM；
    和/或，流动相的流速为0.5～1.5ml/min；
    和/或，色谱柱为C 18色谱柱；
    和/或，色谱柱的柱温为24～26℃。
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