CN112379028A - Method for rapidly detecting content of paclitaxel in-vivo nano preparation - Google Patents
Method for rapidly detecting content of paclitaxel in-vivo nano preparation Download PDFInfo
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Abstract
The invention relates to a method for rapidly detecting the concentration of paclitaxel in animal plasma, animal tissues and human plasma after the administration of a paclitaxel nano preparation, which comprises the steps of detecting a treated liquid sample after pretreatment by adopting an ultra-high performance liquid chromatography tandem mass spectrometry UHPLC-MS/MS (ultra high performance liquid chromatography-mass spectrometry), wherein the pretreatment process comprises the steps of adding a biological matrix sample into an acetonitrile solution containing paclitaxel-D5 according to a certain proportion, oscillating and centrifuging, taking out a centrifuged supernatant, and adding pure water according to a certain proportion for dilution to obtain a sample to be detected. The detection method has the characteristics of high reliability, good accuracy, low sample consumption, high sensitivity and high rapid detection flux.
Description
Technical Field
The invention belongs to the field of drug detection, and particularly relates to a method for detecting the content of paclitaxel in a taxus nanoparticle preparation.
Background
Paclitaxel (PTX) is widely used as an anticancer drug in the treatment of numerous solid tumors. However, PTX has poor solubility in water, which greatly limits its further development and application in the field of tumor therapy. To solve this problem, one possible solution is to prepare PTX as a nano-formulation, thereby increasing the solubility of PTX in water. At the same time, the enhanced penetration and retention effects of the tumor tissue may further contribute to the accumulation of PTX nanopreparative agents in the tumor tissue. Therefore, researchers are dedicated to research and develop various low-toxicity and high-efficiency paclitaxel nano-drugs.
The advantages of the nano-drug over small molecule chemotherapeutic drugs are as follows: (1) the solubility of the medicine is improved through the auxiliary materials, and meanwhile, the medicine is protected from being degraded in blood circulation; (2) prolonging the blood circulation time and providing possibility for further increasing the accumulation of the nano-drugs in the tumor; (3) the aggregation of the drug in the tumor tissue is passively or actively enhanced through the EPR effect of the nano-sized material or the introduction of a targeting group; (4) regulating tissue penetration and drug release; (5) reduce the toxic and side effects of the raw medicines and improve the life quality of patients. At present, common paclitaxel nano-drug carriers include paclitaxel liposome, paclitaxel albumin nanoparticles, paclitaxel nano-micelles, paclitaxel-dendrimer, nanocrystals, polymer vesicles, nanogels, exosomes and the like.
Based on low solubility of micromolecule paclitaxel, the traditional paclitaxel preparation needs a cosolvent, so that the curative effect and the safety of the traditional paclitaxel preparation are influenced, the solubility of the paclitaxel liposome, the paclitaxel micelle preparation, the paclitaxel albumin preparation and other dosage forms which are clinically researched and developed are improved to different degrees, the toxic and side effects are reduced, and the characteristics of the several dosage forms are different and have advantages and disadvantages. The paclitaxel concentration of the nano preparation in animal substrates and clinical biological samples is detected in the development process of the nano preparation, and the nano preparation has important significance for describing the motion process of the nano preparation in vivo and clarifying the pharmacokinetic advantages and explaining the toxicology and pharmacodynamic characteristics of the nano preparation. Therefore, the development of a method which can be suitable for rapidly determining the concentration of the paclitaxel in the biological matrix sample after the administration of various nano preparations plays an important role in synergistically promoting the research and development of the paclitaxel nano preparation.
Disclosure of Invention
The invention aims to provide an LC-MS/MS detection method for rapidly detecting the content of paclitaxel in vivo nano-preparations, which comprises the following steps:
s1: sample pretreatment
Collecting a biological matrix sample after paclitaxel nano preparation administration, quantitatively transferring the biological matrix sample to a deep-hole 96-pore plate polypropylene plate, adding the biological matrix sample into acetonitrile solution containing an internal standard, carrying out high-speed centrifugation after oscillation, taking supernate from the sample after high-speed centrifugation, adding ultrapure water according to a certain proportion, diluting and uniformly mixing to obtain a sample to be detected, and carrying out LC-MS/MS analysis;
s2: determination of biological matrix sample to be detected by LC-MS/MS method
I. Chromatographic conditions are as follows:
a chromatographic column: ZORBAX, Eclipse Plus C18 Narrow Bore; column temperature: 20-30 ℃; mobile phase A: an aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile, using A: b, flowing equal gradient elution, wherein the flow rate of a mobile phase is 0.5-1 mL/min;
mass spectrometry conditions:
an ion source: electrospray ionization source ESI; the spraying voltage is 5500V; ion source temperature: 500 ℃; and (4) CUR: 35 psi; scanning mode: positive ion multiple reaction monitoring + MRM.
Preferably, the LC-MS/MS detection method further comprises the steps of:
s3: preparation of biological matrix standard curve sample
Weighing paclitaxel standard substance, dissolving with dimethyl sulfoxide, diluting to constant volume, preparing into stock solution with concentration of about 1mg/mL, diluting with 50% methanol water step by step to obtain standard series working solution, diluting the working solution with different blank biological matrix samples, making into standard curve samples of different biological matrices, pretreating according to the method of the step S1, detecting under the LC-MS/MS condition of the step S2, and drawing paclitaxel standard curve of corresponding biological matrix according to the detection result;
s4: calculation of paclitaxel content in the biological matrix sample to be tested
And (4) determining the peak area ratio of the paclitaxel in the biological matrix to be detected and the internal standard substance according to the LC-MS/MS method in the step S2, substituting the peak area ratio into the paclitaxel standard curve of the corresponding biological matrix drawn in the step S3, and calculating the content of the paclitaxel in the biological matrix sample to be detected.
Preferably, in the sample pretreatment of S1, the paclitaxel nanoformulation is selected from paclitaxel micelle, paclitaxel cell nanoformulation, paclitaxel nanocrystal, paclitaxel albumin nanoparticle, or paclitaxel liposome.
Preferably, the paclitaxel micelle is a paclitaxel PEG-PLA block copolymer micelle.
Preferably, in the sample pretreatment of S1, the bio-matrix sample is a plasma sample or a tissue homogenate sample.
Preferably, in the sample pretreatment of S1, the volume ratio of the bio-matrix sample to the acetonitrile solution containing the internal standard is 1: 2-1: 10; more preferably, the volume ratio of the biological matrix sample to the acetonitrile solution containing the internal standard is 1: 5-1: 10; most preferably, the volume ratio of the bio-matrix sample to the acetonitrile solution containing the internal standard is 1: 5.
Preferably, in the sample pretreatment of S1, the dilution and mixing ratio of the supernatant to ultrapure water is 1: 0.5-1: 5; more preferably, the dilution and blending ratio of the supernatant to the ultrapure water is 1: 1.
Preferably, in the sample pretreatment of S1, the acetonitrile solution containing the internal standard is an isotope labeled paclitaxel-D5 solution prepared from acetonitrile and having a concentration of 200 ng/mL.
Preferably, in the chromatographic conditions of S2, the column temperature is 25 ℃ and the flow rate of the mobile phase is 0.6 mL/min.
Preferably, in the chromatographic conditions of S2, a volume ratio of 45: 55A: b flow equal gradient elution.
Preferably, under the mass spectrometry conditions of S2, paclitaxel and paclitaxel-D5 react with m/z 854.5 → 286.1 and m/z859.5 → 291.0 respectively, and the collision energy CE is 22 and 25eV respectively.
According to the description of the scheme, the beneficial effects of the invention are as follows:
1. the invention combines a sample pretreatment method and an ultra-high performance liquid chromatography-tandem mass spectrometry detection method, and establishes a method which can be simultaneously suitable for detecting the concentration of paclitaxel in a plasma sample or a tissue homogenate sample after administration of various nano preparations such as paclitaxel micelles, paclitaxel cell nano preparations, paclitaxel nanocrystals, paclitaxel albumin nanoparticles, paclitaxel liposomes and the like.
2. The invention adopts a pretreatment method which is easy to operate and suitable for extracting the taxol nanometer preparation, can remove sample impurities, reduce matrix effect and extract the taxol in different nanometer preparations, and the pretreatment method has simple and rapid operation and high flux.
3. The method adopts UHPLC-MS/MS to detect the concentration of the paclitaxel in the biological matrix, and by optimizing the detection method, the retention time of the paclitaxel and the internal standard is 0.75min, the total analysis time is 2min, the total analysis time is short, the analysis efficiency is high, and the chromatographic peak type is good. The analysis method can meet the requirements of the guiding principle in the biological sample detection regulation through methodology verification.
Drawings
FIG. 1 is the ion scanning mass spectrum of paclitaxel product in LC-MS/MS detection method.
FIG. 2 is the ion scanning mass spectrum of paclitaxel-D5 in the LC-MS/MS detection method.
FIG. 3 is an MRM chromatogram of LC-MS/MS for detecting paclitaxel (left) and paclitaxel-D5 (right) in human blank plasma (A) and lower limit of quantitation (B) samples.
FIG. 4 is a typical standard curve diagram of LC-MS/MS measurement of paclitaxel concentration in human plasma.
FIG. 5 is a chromatogram of paclitaxel determined by LC-MS/MS after adding precipitants in different proportions to a sample of a biological matrix, wherein A is 50. mu.L: 100. mu.L, B is 50. mu.L: 150. mu.L, C is 50. mu.L: 250. mu.L, and D is 50. mu.L: 500. mu.L.
FIG. 6 is a graph of the mean concentration of paclitaxel in blood versus time after intravenous drip of 4 doses of the paclitaxel micelle nano-formulation in a patient.
FIG. 7 is a graph showing the tissue distribution of paclitaxel in different tissues after intravenous administration of 5mg/kg paclitaxel albumin nanoparticle formulation to tumor-bearing mice.
FIG. 8 is a graph of mean concentration of paclitaxel in blood versus time for rats intravenously injected with 5mg/mL paclitaxel injection and paclitaxel cell nano-formulations.
FIG. 9 is a graph of the concentration of paclitaxel in blood versus time pharmacokinetics after gavage administration of 10mg/kg of the paclitaxel nanocrystal formulation to rats.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
Example 1 establishment of a method for determining paclitaxel in human plasma by LC-MS/MS
1. Preparation of standard sample and related solution
1.1 Standard simulation samples
Accurately weighing a proper amount of paclitaxel reference substances, dissolving with dimethyl sulfoxide, fixing the volume, accurately sucking a proper amount of respective stock solutions, and adding methanol: diluting the standard series working solution with water (1:1, v/v) step by step to prepare a simulated biological sample with the paclitaxel concentration range of 10.0-1000 ng/mL, wherein the simulated biological sample is used for drawing a standard curve.
1.2 quality control of samples
Preparing paclitaxel quality control samples with three concentration levels by a method similar to that of standard series samples, wherein the low quality control concentration is 25ng/mL, the medium quality control concentration is 500ng/mL, and the high quality control concentration is 7500 ng/mL;
1.3 internal standard solution
Quantitatively weighing 1mg of paclitaxel-D5 reference substance, dissolving with dimethyl sulfoxide and fixing the volume to prepare paclitaxel-D5 internal standard stock solution with the concentration of 200 mug/mL, precisely sucking a proper amount of the internal standard stock solution, and adding acetonitrile for dilution to obtain the paclitaxel-D5 internal standard acetonitrile solution with the concentration of 200 ng/mL.
2. Sample pretreatment
In clinical tests, after a patient is injected with a paclitaxel micelle preparation (paclitaxel micelle formed by paclitaxel self-assembly loaded by PEG-PLA block polymer), peripheral venous blood is collected for about 2.5mL respectively at 0h, 0.25h, 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 12h, 24h (D2), 48h (D3) and 72h (D4) after administration, and separated plasma is frozen and stored until pretreatment analysis is carried out.
Quantitatively transferring each plasma sample into a deep-hole 96-well plate polypropylene plate with 50 mu L to 2.0mL, adding 250 mu L of the acetonitrile solution containing the paclitaxel-D5 internal standard, oscillating, performing high-speed centrifugation, taking supernate from the sample subjected to high-speed centrifugation, adding ultrapure water according to the volume ratio of 1:1, diluting and uniformly mixing to obtain a sample to be detected, and performing LC-MS/MS analysis.
3. Analytical instrument and detection conditions
3.1, analytical instruments
LC-30AD ultra-high liquid chromatography system, Shimadzu corporation, Japan, and SCIEX Triple Quad 5500 Triple quadrupole tandem mass spectrometer, AB Sciex corporation, equipped with an electrospray ionization source (ESI).
3.2 chromatographic conditions
A chromatographic column: ZORBAX, Eclipse Plus C18 Narrow Bore, 2.1X 50mm, 5 μm (Agilent); column temperature: 25 ℃; sample introduction volume: 2 mu L of the solution; mobile phase A: an aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile in a volume ratio of 45: 55A: b, equal gradient elution, the temperature of an automatic sample injector is 5 ℃, and the flow rate of a mobile phase is 0.6 mL/min; the needle wash was 50% aqueous methanol, and was washed for 5 seconds before and after aspiration.
3.3 Mass Spectrometry conditions
An ion source: electrospray ionization source ESI; the spraying voltage is 5500V; ion source temperature: 500 ℃; and (4) CUR: 35 psi; scanning mode: positive ion multiple reaction monitoring + MRM, paclitaxel and paclitaxel-D5 ion reactions were m/z 854.5 → 286.1 and m/z859.5 → 291.0, respectively, and collision energy CE was 22 and 25eV, respectively.
4. Methodology validation
The methodology of the method is verified according to Chinese pharmacopoeia and American FDA guiding principles, and the contents comprise stability, selectivity, linearity, accuracy, precision, residual effect, recovery rate, matrix effect and micelle plasma sample determination accuracy.
4.1, selectivity
Processing six blank plasmas from different sources and the respectively prepared quantitative lower limit samples, then carrying out sample injection analysis to obtain MRM chromatograms of paclitaxel in the blank plasma samples and the quantitative lower limit samples, and comparing the blank chromatograms to obtain that the peak areas of the interferents are all less than 20% of the peak area of the quantitative lower limit object to be detected and less than 5% of the peak area of the internal standard, as shown in figure 3.
4.2, precision and accuracy
The method verifies and analyzes six samples of each quality control sample for measuring three concentrations in each batch, continuously measures three batches, calculates the precision and accuracy between the batches, and the precision between the batches of the QC samples of each concentration level is less than 15 percent, and the accuracy deviation is between +/-15 percent.
4.3, Standard Curve
The physical concentration to be measured is used as an abscissa (x), the peak area ratio of the substance to be measured to the internal standard substance is used as an ordinate (y), regression operation is carried out by using a weighted (W is 1/x2) least square method, the obtained linear regression equation is a standard curve, the verification result shows that the linear relation of the paclitaxel in the concentration range of 10.0-1000 ng/mL is good, and a typical linear graph of the standard curve is shown in FIG. 4.
4.4 residual Effect
The residual effect is verified by sampling a blank sample after the detection of the high-concentration sample, and analyzing the response value of the instrument at the time of paclitaxel and internal standard peak appearance. And after the upper limit sample is quantified, sampling blank plasma samples, wherein the chromatographic peak areas at the retention time of paclitaxel of the blank samples are all less than 20% of the quantitative lower limit peak area of the standard curve on the day, and the chromatographic peak areas at the retention time of the internal standard samples are all less than 5% of the quantitative lower limit internal standard peak area of the standard curve on the day.
4.5, matrix Effect
Taking blank plasma of 6 different sources to prepare low, medium and high concentration quality control plasma samples, taking pure water as a reference to prepare low, medium and high concentration reference samples, carrying out the same treatment according to a sample pretreatment item, and respectively calculating the matrix effect of each matrix sample analyte, the matrix effect mean value of the same concentration analyte, and the mean value and CV% of the matrix effects of all the matrix samples with different concentrations by taking the reference sample peak area mean value as 100%. And calculating the matrix effect of the internal standard peak area mean value and the internal standard matrix effect mean value of all the matrixes according to the matrix source. In the blood plasma of 6 different sources, the internal standard normalized matrix factor% CV of 6 individual blood plasma matrixes at 3 concentration levels is less than or equal to 15.0%, and the matrix effect result meets the acceptance standard.
4.6 extraction recovery
Preparing low, medium and high concentration quality control plasma samples, analyzing 6 samples at each concentration, taking blank plasma, carrying out the same treatment except that an internal standard is not added, adding a control solution with a certain concentration into the obtained supernatant to ensure that the final concentrations of the object to be detected and the internal standard are respectively the same as the theoretical concentrations of the low, medium and high quality control samples after treatment, analyzing 6 samples at each concentration, calculating the recovery rate by using the peak area of two treatment methods at each concentration, and ensuring that the recovery rate of the low, medium and high concentrations of paclitaxel is 85.6-95.2%. The extraction recovery of the internal standard was 98.9%.
4.7 paclitaxel micelle plasma sample application accuracy
Taking a paclitaxel micelle preparation, diluting a paclitaxel micelle solution to the concentration of QC working solution by taking water as a diluent, adding plasma to prepare paclitaxel micelle plasma samples with high, medium and low quality control concentrations, repeatedly preparing 6 samples at each concentration, processing according to a sample pretreatment method, then carrying out sample injection analysis, and calculating the accuracy of the paclitaxel micelle plasma samples by using a standard curve prepared from paclitaxel solution carried in an analysis batch.
And calculating the accuracy deviation and RSD of the paclitaxel micelle plasma QC sample according to the analyte standard curve of the paclitaxel micelle plasma samples with 3 concentration levels, wherein the accuracy deviation and the RSD both accord with the acceptance standard.
4.8, stability
The stability of the paclitaxel samples during sample collection, storage and analysis was verified, with the results shown in table 1, indicating that the paclitaxel samples were substantially stable under the conditions shown in the table below.
TABLE 1 stability of paclitaxel stock, working fluid and plasma samples
Example 2 optimization of the ratio of the Biomatrix sample to the precipitating agent
Preparing a plasma quality control sample according to the method described in example 1, quantitatively transferring 50 μ L to 2.0mL of deep-hole 96-well plate polypropylene plate for each plasma sample, respectively adding 100 μ L, 150 μ L, 250 μ L and 500 μ L of acetonitrile precipitant containing taxol-D5 internal standard (with the concentration of 200ng/mL), oscillating, centrifuging at high speed, taking the supernatant of the sample after the high speed centrifugation, adding ultrapure water according to the volume ratio of 1:1, diluting and mixing uniformly to obtain a sample to be tested, performing LC-MS/MS analysis according to the conditions of example 1, and obtaining the chromatogram of the taxol as shown in FIG. 5.
In terms of the amount of the precipitant, as shown in tables 3-4, after the precipitant is added in a ratio of 1:2, the matrix effect and the extraction recovery rate of the QC samples with different concentrations are lower than 80%, and after the precipitant is added in a ratio of 1:3, the matrix effect and the extraction recovery rate of the QC samples with different concentrations are lower than 85%, 83.7% -87.6%, and the results of the matrix effect and the extraction recovery rate are not ideal. When the precipitant is added according to the ratio of 1:5 to 1:10, the matrix effect and the extraction recovery rate of QC samples with different concentrations are both higher than 90%. Further analyzing the chromatographic peak results of adding the precipitating agents with different ratios, when the precipitating agents are added according to the ratio of 1:2 and 1:3, the chromatographic peak of part of samples has a shoulder peak, and when the precipitating agents are added according to the ratio of 1:10, the chromatographic peak tip of the samples is cracked. Since shoulder seams and peak tip cracks in chromatographic peaks are not beneficial to the correct integration of chromatographic peak areas and the accurate calculation of subsequent paclitaxel concentration, the effect of the precipitant with the ratio is inferior to that with the ratio of 1: 5. Therefore, the results of peak shape of chromatographic peak, sample residue, matrix effect, extraction recovery were evaluated in combination, and the volume ratio of 1:5 of the bio-matrix sample to the precipitant containing the internal standard was determined as the optimum volume ratio.
TABLE 3 matrix Effect of plasma QC samples with different ratios of precipitant addition
TABLE 4 recovery and residual rate of plasma QC samples after adding precipitants in different proportions
Example 3 detection of the concentration of paclitaxel in plasma after patient injection with paclitaxel micelles
1. Dosing regimens
Intravenous drip administration, intravenousThe infusion time was 3h, and 5 dose groups (175 mg/m) were designed2、225mg/m2、300mg/m2、390mg/m2、435mg/m2) The paclitaxel micelle formulation of (1) was subjected to a dose-finding study.
2. Sample collection
Blood is collected for 0h, 0.25h, 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 12h, 24h (D2), 48h (D3) and 72h (D4) after the instillation is finished, about 2mL of peripheral venous blood is collected at each point, after the sampling, the blood is centrifuged at the speed of 2000 Xg for 10 minutes at the temperature of 4 ℃, and separated plasma samples are frozen and stored until the detection.
3. Concentration detection
Referring to the pretreatment and detection methods of example 1 of the present application, the mean time course of each dose group is shown in fig. 6, which is a graph showing the paclitaxel concentration in human plasma after phase 1 clinical injection of paclitaxel. The pharmacokinetic parameters calculated after administration of five dose groups of paclitaxel micelle injections are shown in table 2.
TABLE 2 plasma pharmacokinetic parameters calculated after administration of paclitaxel micelles for five dose groups
Example 4 detection of paclitaxel concentration in plasma and tissue after administration of paclitaxel albumin nanoparticles
1. Dosing regimens and sample collection
Tumor-bearing mice with tumors growing to 100mm3 were taken, 6 mice per time point. The paclitaxel albumin nanoparticle preparation is administered by tail vein injection of tumor-bearing mice, the dose is 5mg/kg, and the heart, liver, spleen, lung, kidney and tumor are taken out at 0.5h, 2h and 8h after administration, the blood is cleaned by normal saline, and the filter paper is sucked dry. The distribution of paclitaxel in tumor-bearing mice was determined by sample injection analysis with reference to the sample treatment method and LC-MS/MS detection method described in example 1 above.
2. Concentration detection
After the paclitaxel albumin nanoparticle preparation is administrated, the method is used for measuring the concentration of paclitaxel in blood plasma, heart, liver, spleen, lung, kidney and tumor tissues of mice. The results of the detection of paclitaxel concentration in different tissues after intravenous administration of 5mg/kg paclitaxel albumin nanoparticle preparation to tumor-bearing mice are summarized in FIG. 7.
Example 5 detection of paclitaxel concentration in blood after paclitaxel cell Nanodiment administration
12 SD male rats with the body weight of about 200g are selected, the male rats and the female rats are half of the SD male rats, before animal experiments, the SD male rats are randomly divided into 2 groups which are respectively a paclitaxel injection group or a paclitaxel neutrophilic granulocyte nano-preparation group (the preparation method is shown in CN201711106703.2), and each group comprises 6 rats. Tail vein injection administration is carried out, the administration dose is 5mg/kg, and the administration is carried out according to the preset time points: 10min, 30min, 45min, 1.5h, 3h, 6h, 10h and 24h, blood was taken from the jugular vein of the rat, the blood sample was cryopreserved, the pretreatment of the blood sample of the rat was performed according to the method of example 1, the content of paclitaxel drug in the blood sample was analyzed and a pharmacokinetic curve was drawn under the LC-MS/MS analysis conditions of example 1, and the mean concentration-time pharmacokinetic curve of paclitaxel in the blood after the rat was intravenously injected with paclitaxel injection and paclitaxel cell nano-formulation is shown in fig. 8.
Since the nano-preparation of this embodiment is a paclitaxel cell preparation prepared from paclitaxel cells, if conventional pharmacokinetic studies are employed, plasma or serum is centrifuged after animal administration, most of the paclitaxel cell preparation is centrifuged to the lower layer, and the concentration of paclitaxel in plasma is severely low and cannot represent the actual paclitaxel concentration in blood circulation, so that a blood sample is required for pharmacokinetic studies. The treatment by the pretreatment method of the application example 1 can realize the accurate determination of the concentration of the paclitaxel in the blood of the rat.
Example 6 detection of paclitaxel concentration in plasma after oral administration of paclitaxel nanocrystals
Rats were fasted for at least 12 hours before dosing in 5 rats, had free access to water and were fed 4h after dosing. After the 10mg/kg paclitaxel nanocrystal formulation was gavaged in rats, blood samples were collected through the retroorbital venous plexus approximately 15min, 30min, 1, 2, 4, 6, 8, 10 and 24H after administration, each blood sample was collected at about 0.3mL to contain 5. mu.L of 5% K2 EDTA.2Ht2O (national chemical group chemical agent Co., Ltd.) antibodyA centrifuge tube of a coagulant. Temporarily storing the collected blood on ice for no more than 30min until centrifugation, centrifuging the blood (at 2-8 ℃ and 8000rpm for 5min), collecting plasma into a 96-hole sample plate, and transferring to a refrigerator at-15 ℃ or below for temporary storage. And after the animal experiment is completed, transferring the plasma sample to a biological sample library, and storing at the temperature of less than or equal to-65 ℃ until the plasma sample is to be detected.
The method is pre-processed according to the method in example 1 of the application and is successfully applied to the determination of the concentration of the paclitaxel in the plasma of rats after the oral administration of the paclitaxel nanocrystal preparation, and the result of the determination of the concentration of the paclitaxel in the plasma of rats after the application of the method to the administration of the paclitaxel nanocrystals is accurate and reliable. The pharmacokinetic profile of paclitaxel concentration in blood versus time after gastric gavage of 5 rats given 10mg/kg of paclitaxel nanocrystal formulation is shown in FIG. 9.
The above examples prove that the detection method of the invention can quantitatively analyze the paclitaxel concentration in various biological matrixes of different species after administration of various paclitaxel nano preparations, and has the advantages of wide detection trial range, good accuracy and low sensitivity on the paclitaxel concentration in the biological matrixes.
It is to be understood that the above-described embodiments of the present invention are merely illustrative of or explaining the principles of the invention and are not to be construed as limiting the invention. Therefore, any modification, equivalent replacement, improvement and the like made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An LC-MS/MS detection method for rapidly detecting the content of paclitaxel in vivo nano-preparations is characterized by comprising the following steps:
s1: sample pretreatment
Collecting a biological matrix sample after paclitaxel nano preparation administration, quantitatively transferring the biological matrix sample to a deep-hole 96-pore plate polypropylene plate, adding the biological matrix sample into acetonitrile solution containing an internal standard, carrying out high-speed centrifugation after oscillation, taking supernate from the sample after high-speed centrifugation, adding ultrapure water according to a certain proportion, diluting and uniformly mixing to obtain a sample to be detected, and carrying out LC-MS/MS analysis;
s2: determination of biological matrix sample to be detected by LC-MS/MS method
I. Chromatographic conditions are as follows:
a chromatographic column: ZORBAX, Eclipse Plus C18 Narrow Bore; column temperature: 20-30 ℃; mobile phase A: an aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile, using A: b, flowing equal gradient elution, wherein the flow rate of a mobile phase is 0.5-1 mL/min;
mass spectrometry conditions:
an ion source: electrospray ionization source ESI; the spraying voltage is 5500V; ion source temperature: 500 ℃; and (4) CUR: 35 psi; scanning mode: positive ion multiple reaction monitoring + MRM.
2. The LC-MS/MS detection method according to claim 1, wherein said detection method further comprises the steps of:
s3: preparation of biological matrix standard curve sample
Weighing paclitaxel standard substance, dissolving with dimethyl sulfoxide, diluting to constant volume, preparing into stock solution with concentration of about 1mg/mL, diluting with 50% methanol water step by step to obtain standard series working solution, diluting the working solution with different blank biological matrix samples, making into standard curve samples of different biological matrices, pretreating according to the method of the step S1, detecting under the LC-MS/MS condition of the step S2, and drawing paclitaxel standard curve of corresponding biological matrix according to the detection result;
s4: calculation of paclitaxel content in the biological matrix sample to be tested
And (4) determining the peak area ratio of the paclitaxel in the biological matrix to be detected and the internal standard substance according to the LC-MS/MS method in the step S2, substituting the peak area ratio into the paclitaxel standard curve of the corresponding biological matrix drawn in the step S3, and calculating the content of the paclitaxel in the biological matrix sample to be detected.
3. The LC-MS/MS detection method of claim 1 or 2, wherein in the sample pretreatment of S1, the paclitaxel nano-preparation is selected from paclitaxel micelle, paclitaxel cell nano-preparation, paclitaxel nanocrystal, paclitaxel albumin nanoparticle, or paclitaxel liposome.
4. The LC-MS/MS detection method of claim 1 or 2, wherein in the sample pre-treatment of S1, the bio-matrix sample is a plasma sample or a tissue homogenate sample.
5. The LC-MS/MS detection method according to claim 1 or 2, wherein in the sample pretreatment of S1, the volume ratio of the bio-matrix sample to the acetonitrile solution containing the internal standard is 1: 2-1: 10; more preferably, the volume ratio of the biological matrix sample to the acetonitrile solution containing the internal standard is 1: 5-1: 10; most preferably, the volume ratio of the bio-matrix sample to the acetonitrile solution containing the internal standard is 1: 5.
6. The LC-MS/MS detection method according to claim 1 or 2, wherein in the sample pretreatment of S1, the dilution and mixing ratio of the supernatant to ultrapure water is 1: 0.5-1: 5; more preferably, the dilution and blending ratio of the supernatant to the ultrapure water is 1: 1.
7. The LC-MS/MS detection method of claim 1 or 2, wherein in the sample pretreatment of S1, the acetonitrile solution containing the internal standard is an isotope labeled paclitaxel-D5 solution prepared with acetonitrile at a concentration of 200 ng/mL.
8. The LC-MS/MS detection method according to claim 1 or 2, wherein in the chromatographic conditions of S2, the column temperature is 25 ℃ and the flow rate of the mobile phase is 0.6 mL/min.
9. The LC-MS/MS detection method according to claim 1 or 2, characterized in that in the chromatographic conditions of S2, a volume ratio of 45: 55A: b flow equal gradient elution.
10. The LC-MS/MS detection method according to claim 1 or 2, wherein under the mass spectrometry condition of S2, paclitaxel and paclitaxel-D5 ion reactions are m/z 854.5 → 286.1 and m/z859.5 → 291.0, respectively, and the collision energy CE is 22 and 25eV, respectively.
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