CN114487157A - Method for separating free-state and liposome-state eribulin in biological matrix - Google Patents
Method for separating free-state and liposome-state eribulin in biological matrix Download PDFInfo
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- CN114487157A CN114487157A CN202111592700.0A CN202111592700A CN114487157A CN 114487157 A CN114487157 A CN 114487157A CN 202111592700 A CN202111592700 A CN 202111592700A CN 114487157 A CN114487157 A CN 114487157A
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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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
- B01D15/424—Elution mode
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The invention discloses a method for separating free-state and liposome-state eribulin in a biological matrix, which comprises the steps of transferring the free-state and liposome-state eribulin in the biological matrix to a solid-phase extraction column, leaching and eluting the solid-phase extraction column, and separating the free-state and liposome-state eribulin in the biological matrix. The invention achieves the purpose of separating free eribulin and liposome eribulin in the biological matrix based on selecting a solid-phase extraction column with proper filler, an optimal leaching reagent and an elution reagent, and finally can support the quantitative detection of the free eribulin and the total eribulin in the biological matrix. By using the method, the recovery rate of the free liposome can reach more than 90 percent, and the requirement of quantitative detection is basically met.
Description
Technical Field
The invention relates to a method for separating free eribulin and liposome eribulin in a biological matrix, in particular to research on quantitative detection of free eribulin and total eribulin in a living body in preclinical and clinical experiments, and belongs to the technical field of biology.
Background
Eribulin produces cytotoxicity by binding to the vinka alkaloid binding domain, which in turn causes globular tubulin aggregation, resulting in the arrest of the mitotic process and cell death. Efficacy testing has demonstrated that eribulin is therapeutically active against many types of tumors, such as breast cancer, urothelial tumors, prostate cancer, and non-small cell lung cancer, including patients previously treated with multiple anti-microtubule drugs. The dose-limiting toxicities of eribulin were mainly myelosuppression and neutropenia, but also included other non-hematologic toxicity events including fatigue, nausea, hair loss. The reduction of eribulin toxicity, the reduction of dosing frequency, and the improvement of patient compliance are important directions in promoting the development of eribulin.
Liposomes (liposomes) are an artificial membrane. The hydrophilic head of phospholipid molecules in water is inserted into the water, the hydrophobic tail of the liposome extends to the air, and the spherical liposome with double-layer lipid molecules is formed after stirring, and the diameter is different from 25-1000 nm. The liposome can be used for transgenosis or preparing medicines, and the medicines are delivered into cells by utilizing the characteristic that the liposome can be fused with cell membranes. The liposome is used for wrapping the eribulin, so that the aim of reducing toxicity can be achieved, the half-life period of the drug in a human body is prolonged, and the administration frequency is effectively reduced.
By utilizing liposome administration, free drugs are components which directly exert drug effects or toxicity, and quantitative detection of the free components in the biological matrix can more intuitively reflect the relationship between the drug effects and the pharmacokinetics and between the toxicity and the pharmacokinetics. The existing method for separating free eribulin is mainly an ultra-high-speed centrifugation method. The analysis of free eribulin was carried out by centrifugation at 355040g for 4 hours. The method has very high requirements on the centrifuge, general laboratories have no conditions, and the sample processing time is very long, so the method is not suitable for large-batch clinical \ preclinical tests. The existing experiment greatly needs a method for quickly and conveniently separating free eribulin.
Disclosure of Invention
The invention aims to provide a method for separating eribulin in a free state and a liposome state in a biological matrix so as to meet the quantitative detection requirement of the eribulin in the free component in the biological matrix, and the method is simple and rapid to operate and high in operability.
In order to achieve the purpose, the invention provides a method for separating free-state and liposome-state eribulin in a biological matrix, which comprises the steps of transferring the free-state and liposome-state eribulin in the biological matrix to a solid-phase extraction column, leaching and eluting the solid-phase extraction column, and separating the free-state and liposome-state eribulin in the biological matrix.
Further, the solid phase extraction column is a polyvinylpyrrolidone solid phase extraction column.
Further, the elution adopts a pure water eluent, and the elution adopts methanol, isopropanol or acetonitrile eluent.
Further, the volume of the pure water eluting agent is 0.8-1.2mL, and the volume of the eluent is more than or equal to 0.3 mL.
Further, the solid phase extraction column specifications were 30mg/1mL, 60mg/3mL, 150mg/6mL, 200mg/6mL, and 500mg/6 mL.
Further, the solid phase extraction column is equilibrated with not less than 0.1mL of plasma before loading and before rinsing, respectively.
Further, the biological matrix is one of plasma, serum, whole blood, feces, and tissue homogenate.
Further, the method specifically comprises the following steps: firstly, 1mL of 2-8 ℃ precooled methanol activated solid phase extraction column → 1mL of 2-8 ℃ precooled ultrapure water activated solid phase extraction column → 0.1mL of 2-8 ℃ precooled plasma balanced solid phase extraction column environment → 0.1mL of biological matrix samples of free eribulin and liposome eribulin are transferred to the solid phase extraction column → after the samples are completely absorbed by the solid phase extraction column, 0.1mL of 2-8 ℃ precooled plasma is used for balancing → 0.5mL of 2-8 ℃ ultrapure water is used for rinsing the solid phase extraction column for 2 times → 0.3mL of 2-8 ℃ precooled methanol is used for eluting the solid phase extraction column for 2 times, eluent is combined and collected → the eluent is dried by nitrogen under the condition of room temperature, 0.3mL of 20% acetonitrile containing 0.1% of FA is used for redissolving, and 2 μ L of acetonitrile is transferred to a liquid mass spectrometer for quantitative analysis.
The invention achieves the following beneficial effects:
(1) the method selects a specific solid phase extraction column and proper leaching and eluting reagents. The different adsorption capacities of the solid-phase extraction column to the liposome and the free eribulin are utilized to achieve the purpose of separating the free eribulin and the liposome eribulin. By using the method, the recovery rate of the free liposome can reach more than 90 percent, and the requirement of quantitative detection is basically met.
(2) The invention is a brand new method for separating free eribulin and liposome eribulin, can meet the research requirements of pharmacokinetics and toxicokinetics in preclinical/clinical trials of eribulin, and meets the regulations of guiding principles. The invention is not only suitable for blood plasma and whole blood, but also suitable for various biological matrixes such as excrement, tissue homogenate and the like.
(3) Compared with other existing separation methods, the method has the advantages that test consumables and instruments are easy to obtain, operation is simple and safe, batch operation can be achieved, and the requirement for large-batch detection is met.
Drawings
Figure 1 is the standard curve accuracy in example 5.
Detailed Description
The present invention will be further described with reference to the following examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
The reagents and instruments adopted in the invention are all common in the market and can be purchased from the market.
Example 1
Firstly, 1mL of 2-8 ℃ precooled methanol activated solid-phase extraction column → 1mL of 2-8 ℃ precooled ultrapure water activated solid-phase extraction column → 0.1mL of 2-8 ℃ precooled plasma to improve the solid-phase extraction column environment → 0.1mL of a biological matrix sample containing 1.0 muL/mL of free eribulin is transferred to the solid-phase extraction column → after the sample is completely absorbed by the solid-phase extraction column, 0.1mL of 2-8 ℃ precooled plasma is used for carrying out initial leaching on the sample → 0.5mL of 2-8 ℃ precooled ultrapure water is used for leaching the solid-phase extraction column for 2 times → 0.3mL of 2-8 ℃ acetonitrile is used for carrying out elution on the solid-phase extraction column for 4 times, eluent is respectively collected → 4 parts of eluent are dried by nitrogen under the condition of room temperature, 0.3mL of 20% acetonitrile containing 0.1% of FA is respectively used for redissolution, and 2 muL is transferred to a liquid mass spectrometer for quantitative analysis.
The above procedure was repeated and the sample concentration was changed to 2.0. mu.L/mL. The results are shown in Table 1.
Example 2
The elution reagent was replaced by isopropanol instead of acetonitrile on the basis of example 1. The results are shown in Table 1.
Example 3
The elution reagent was replaced by methanol instead of acetonitrile on the basis of example 1. The results are shown in Table 1.
TABLE 1 elution efficiency of different eluents
By comparing the results of different eluents in table 1, when the sample concentration is 1 mug/mL, 3 eluents can elute more than 80% after 2 times of elution, but when the sample concentration is changed to 2 mug/mL, the elution effect of acetonitrile is obviously reduced, only 36% is eluted after 2 times, the elution ratio of isopropanol and methanol can still reach more than 80% after 2 times of elution, and the elution ratio is not changed along with the change of the sample concentration. Therefore, methanol and isopropanol can be used as the eluent of the invention. Further considering that the elution rate of methanol for 2 times can reach more than 95%, methanol is selected as the preferred eluent of the invention.
Example 4
Firstly, 1mL of 2-8 ℃ precooled methanol activated solid-phase extraction column → 1mL of 2-8 ℃ precooled ultrapure water activated solid-phase extraction column → 0.1mL of 2-8 ℃ precooled plasma to improve the solid-phase extraction column environment → 0.1mL of preparation containing 0.75mg/mL liposome eribulin is transferred to the solid-phase extraction column → after the sample is completely absorbed by the solid-phase extraction column, the sample is subjected to primary leaching by 0.1mL of 2-8 ℃ precooled plasma → the solid-phase extraction column is leached by 0.5mL of 2-8 ℃ precooled ultrapure water for 2 times → the solid-phase extraction column is eluted by 0.3mL of 2-8 ℃ precooled methanol for 2 times, eluent is combined and collected → the eluent is dried by nitrogen under the condition of room temperature, and is redissolved by 0.3mL of 20% acetonitrile containing 0.1% of FA, and 2 μ L of the eluent is transferred to a liquid chromatograph-mass spectrometer for quantitative analysis. The replicates were repeated 3 times. The results are shown in Table 2.
At the same time, 0.1mL of the formulation containing 0.75mg/mL liposomal eribulin was added to the EP tube, 0.2mL acetonitrile was added, and the liposomes were demulsified by vortexing vigorously for 5 minutes. Transfer 10. mu.L to an EP tube containing 990. mu.L of 20% acetonitrile containing 0.1% FA, mix well and transfer 2. mu.L to LC-MS for quantitative analysis. The replicates were repeated 3 times. The results are shown in Table 2.
TABLE 2 Selectivity of the solid phase extraction method
The results in table 2 show that the separation of the eribulin liposome preparation by the method of the present invention can detect the free result, and the total amount of the preparation is only 0.30%, which is far lower than the pharmacopeia requirement (20%), so the selectivity of the present invention completely meets the detection requirement.
Example 5
According to the general requirements of pharmacopoeia 9012, a standard curve sample is prepared as a quality control sample, and the precision and accuracy data are collected by adopting the method. The precision and accuracy results are shown in Table 3, and the standard curve accuracy results are shown in FIG. 1.
Table 3 density and accuracy results
The results of table 3 and fig. 1 show that the precision and accuracy of the results of the method meet the requirements of general rules of pharmacopoeia 9012.
Example 6
2 Beagle dogs are selected to be injected with the eribulin liposome intravenously, and blood is collected immediately after administration, 5min, 15min, 30min, 2h, 6h, 10h and 24 h. Centrifuging whole blood, packaging into two parts, storing at 2-8 deg.C for detecting free eribulin in plasma, and storing at-60 deg.C for detecting total eribulin. The results are shown in Table 4.
TABLE 4 free and Total Eribulin concentrations in Beagle canine plasma
The results in table 4 are in line with the metabolic trends of total and free eribulin in animals after eribulin liposomes were administered, and the method meets the needs of the experiment.
To sum up the results of examples 4-6, the present invention can meet the new requirements for quantitative detection of free eribulin in preclinical and clinical trials of eribulin liposomes.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A method for separating free-state and liposome-state eribulin in a biological matrix is characterized in that the free-state and liposome-state eribulin in the biological matrix is transferred to a solid-phase extraction column, and is separated after elution and elution.
2. The method of claim 1, wherein the solid phase extraction column is a polyvinylpyrrolidone solid phase extraction column.
3. The method for separating eribulin in free form and in liposomal form in a biological matrix, as claimed in claim 1, wherein said elution is with a pure water eluent, and said elution is with an eluent of methanol, isopropanol, or acetonitrile.
4. The method for separating eribulin in free form and in liposome form in a biological matrix, as claimed in claim 3, wherein said volume of pure water eluent is 0.8-1.2mL, and said volume of eluent is 0.3mL or more.
5. The method of claim 1, wherein the solid phase extraction column is 30mg/1mL, 60mg/3mL, 150mg/6mL, 200mg/6mL and 500mg/6mL in size.
6. The method of claim 1, wherein the column is equilibrated with not less than 0.1mL of plasma for solid phase extraction before loading and before rinsing.
7. The method of separating eribulin in free form and in liposomal form in a biological matrix as claimed in claim 1, wherein said biological matrix is one of plasma, serum, whole blood, fecal matter, and tissue homogenate.
8. The method for separating eribulin in free form and in liposomal form in a biological matrix as claimed in claim 1, further comprising: firstly, 1mL of 2-8 ℃ precooled methanol activated solid phase extraction column → 1mL of 2-8 ℃ precooled ultrapure water activated solid phase extraction column → 0.1mL of 2-8 ℃ precooled plasma balanced solid phase extraction column environment → 0.1mL of biological matrix samples of free eribulin and liposome eribulin are transferred to the solid phase extraction column → after the samples are completely absorbed by the solid phase extraction column, balancing with 0.1mL of 2-8 ℃ pre-cooled plasma → leaching the solid phase extraction column with 0.5mL of 2-8 ℃ pre-cooled ultrapure water for 2 times → eluting the solid phase extraction column with 0.3mL of 2-8 ℃ pre-cooled methanol for 2 times, combining and collecting the eluents → blowing the eluents with nitrogen at room temperature, and reconstituted with 0.3mL of 20% acetonitrile containing 0.1% FA, and transferred 2 μ L to gc for quantitative analysis.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105026398A (en) * | 2013-03-05 | 2015-11-04 | 默克专利股份公司 | Triazolo[4,5-d]pyrimidine derivatives for the treatment of diseases such as cancer |
| CN207440022U (en) * | 2017-10-23 | 2018-06-01 | 苏州国辰生物科技股份有限公司 | A kind of device of the outer free drug of liposome and liposome in separated plasma |
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- 2021-12-23 CN CN202111592700.0A patent/CN114487157A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105026398A (en) * | 2013-03-05 | 2015-11-04 | 默克专利股份公司 | Triazolo[4,5-d]pyrimidine derivatives for the treatment of diseases such as cancer |
| CN207440022U (en) * | 2017-10-23 | 2018-06-01 | 苏州国辰生物科技股份有限公司 | A kind of device of the outer free drug of liposome and liposome in separated plasma |
Non-Patent Citations (3)
| Title |
|---|
| YANKE YU 等: "Characterization of the pharmacokinetics of a liposomal formulation of eribulin mesylate (E7389) in mice", INTERNATIONAL JOURNAL OF PHARMACEUTICS * |
| 周芳: "SPE-HPLC测定紫杉醇脂质体包封率的方法探讨", 黑龙江畜牧兽医 * |
| 熊宁杰 等: "盐酸多柔比星脂质体注射液犬体内药物动力学研究", 沈阳药科大学学报 * |
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