CN107589184B - Analysis and detection method of PEG and PEG-modified drug and application thereof - Google Patents
Analysis and detection method of PEG and PEG-modified drug and application thereof Download PDFInfo
- Publication number
- CN107589184B CN107589184B CN201710706546.2A CN201710706546A CN107589184B CN 107589184 B CN107589184 B CN 107589184B CN 201710706546 A CN201710706546 A CN 201710706546A CN 107589184 B CN107589184 B CN 107589184B
- Authority
- CN
- China
- Prior art keywords
- peg
- detected
- mass
- charge ratio
- collision
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention discloses an analysis and detection method of PEG and PEG drugs, which uses liquid chromatography-quadrupole-time-of-flight mass spectrometry for analysis and detection, separates a substance to be detected containing the PEG and the PEG drugs by liquid chromatography, establishes a TOF MS-Swath continuously variable window, and respectively sets collision energy corresponding to the TOF MS and collision energy corresponding to the Swath; charged ions with different mass-to-charge ratio ranges sequentially enter the collision chamber Q2 by setting a continuously variable window, specific fragments are generated by energy collision of the object to be detected, and the object to be detected is detected by scanning and analyzing the specific fragments. The invention establishes a technical scheme which can simultaneously give consideration to the scanning of the parent ions and the segmented entering of the parent ions into a collision chamber for collision induced dissociation to obtain specific fragment ions, can obtain the distribution of high molecular polymer precursor ions in a specific mass-to-charge ratio interval, and simultaneously completes the qualitative and quantitative analysis of PEG and PEG drugs according to the parent ions and the fragment ions.
Description
Technical Field
The invention belongs to the technical field of drug analysis research, and relates to a method for analyzing PEG and PEG-modified drug biological mass spectrometry.
Background
In recent years, the science and technology of pharmaceutical analysis have been greatly developed to meet the needs of pharmaceutical research and development. In recent years, the rapidly developing liquid chromatography tandem mass spectrometry (LC-MS/MS) technology has provided a possible solution for pegylated drug analysis. Compared with the traditional immunology, HPLC, colorimetric method and the like, the liquid chromatography-tandem mass spectrometry method has great advantages in the aspects of accuracy, precision, selectivity, sensitivity, quantitative dynamic range and the like. At present, mass spectrum quantitative analysis reports of the PEG micromolecular drugs are few, and the main reason is that the PEG protein drugs can adopt a proteomics method to select characteristic peptide fragments after enzymolysis for quantitative analysis; however, no effective enzyme digestion means is available for the PEG micromolecule drug to hydrolyze the PEG micromolecule drug into free drug molecules. In addition, the molecular weight distribution range of the PEGylated drug is wide, even the molecular weight of PEG with the same polymerization degree is not unique, and the LC-MS/MS method based on the scanning mode such as MRM can only carry out quantitative analysis on limited target compounds with determined molecular weight, so that the mass spectrometry analysis of the multi-component polymorphic macromolecular PEGylated drug has great challenge. The in-source fragmentation technique can give rich ion fragment information in the primary mass spectrum. The speed of ions entering a mass spectrum is influenced by utilizing a Declustering Potential (DP), and the higher the Declustering voltage of the taper hole is, the faster the ion speed is, the smaller the ion loss is, and the higher the detection sensitivity is. Too high a cone voltage increases ion-to-ion collisions, causing intra-source fragmentation, resulting in fragment ions. The fragment ions can be used for both the qualitative analysis of the substance and the quantitative analysis by simultaneously monitoring the parent ions and the fragment ions in the MRM scanning mode. By adding larger energy de-clustering voltage (DP) into ion source, PEG and PEG chemical drug are cracked in the ion source to generate specificityFragment ions, namely selecting specific fragment ions for monitoring to complete the quantitative analysis of PEG and PEG drugs; however, since the energy of DP is small, the collision efficiency of CID in source is limited, and this method is not suitable for the analysis of some pegylated drugs with strong binding capacity, especially it is difficult to obtain the specific fragment ions of the pegylated drugs for the drugs. When bonded to a small molecule: (<500) When the molecular weight of PEG above is large (>10K) In the process, the chromatographic behavior of free PEG and PEG drugs is very close, and the PEG and PEG drugs are difficult to separate by chromatography, so that the monitoring of the PEG and the PEG drugs is not suitable only by monitoring specific fragments of the PEG; in addition, the sensitivity of this method is limited because the impact energy of DP is small. MassallThe technology can provide a quantitative method of PEG and PEG drugs to a certain extent, but because of MassallThe technology is that all charged particles enter a collision chamber through Q1 and are smashed into secondary fragments, ions entering the collision chamber have no selectivity, meanwhile, due to the fact that too many ions enter the collision chamber at the same time, the cracking efficiency and the transmission efficiency of primary ions can be obviously reduced, the sensitivity and the selectivity can be limited, meanwhile, due to the fact that chromatographic mass spectrum peak response distribution conditions of PEG or PEG chemical drugs in different mass-to-charge ratio ranges cannot be obtained, and the method is limited in the qualitative analysis capacity of the PEG or PEG chemical drugs.
Disclosure of Invention
The invention aims to solve the technical problem of establishing a technical scheme which can simultaneously give consideration to mother ion scanning and the collision induced dissociation of the segmented mother ions entering a collision chamber to obtain specific fragment ions, can obtain the distribution of high-molecular polymer precursor ions in a specific mass-to-charge ratio range, and simultaneously complete the qualitative and quantitative analysis of PEG and PEG drugs according to the mother ions and the fragment ions. The purpose of the invention is realized by the following technical scheme:
an analysis and detection method of PEG and PEG drugs uses liquid chromatography-quadrupole-time of flight mass spectrometry (LC-Q-Q-TOF MS) to carry out analysis and detection, separates a substance to be detected containing PEG and PEG drugs through liquid chromatography, establishes a TOF MS-Swath continuously variable window, and respectively sets collision energy corresponding to the TOF MS and collision energy corresponding to the Swath; charged ions with different mass-to-charge ratio ranges sequentially enter the collision chamber Q2 by setting a continuously variable window, specific fragments are generated by energy collision of the object to be detected, and the object to be detected is detected by scanning and analyzing the specific fragments.
Further, TOF MS corresponds to a collision energy of 10eV and Swath corresponds to a collision energy of 40 eV.
Further, the channels of the mass-to-charge ratio collection window of the swing are 500-.
Further, the PEG-specific fragment ions selected were at m/z89.0611,133.0869,177.1102,221.1366,265.1622,309.1878,353.2108,397.2359, i.e. 2,3, 4, 5, 6, 7, 8, 9 PEG units.
Further, the detection target in the object to be detected is PEG550, and the distribution window with the highest specific mass-to-charge ratio abundance is m/z 500-700; the detection target in the substance to be detected is PEG750, and the distribution window with the highest specific mass-to-charge ratio abundance is m/z 400-800; the detection target in the object to be detected is PEG2000, and the distribution window with the highest specific mass-to-charge ratio abundance is m/z 600-800; the detection target in the object to be detected is PEG5000, and the distribution window with the highest specific mass-to-charge ratio abundance is m/z 600-800; the detection target in the substance to be detected is PEG2000-Dox, and the distribution window with the highest specific mass-to-charge ratio abundance is m/z 600-1000.
An analysis and detection method of PEG and PEG adriamycin uses liquid chromatogram-quadrupole-time of flight mass spectrometer (LC-Q-Q-TOF MS) to carry out analysis and detection, separates the object to be detected containing PEG and PEG drug by liquid chromatogram, establishes TOF MS-Swath continuously variable window, and respectively sets the collision energy corresponding to TOF MS and the collision energy corresponding to Swath; charged ions with different mass-to-charge ratio ranges sequentially enter a collision chamber Q2 by setting a continuously variable window, a substance to be detected generates specific fragments through energy collision, the substance to be detected is detected through scanning and analyzing the specific fragments, and the detection conditions are as follows:
the chromatographic conditions are as follows: a high performance liquid chromatography system; a chromatographic column: 300SB C18 column, 150mm × 4.6mm I.D., particle size of 5 μm; mobile phase: water containing 0.1% formic acid by volume and acetonitrile containing 0.1% formic acid by volume, and carrying out gradient elution; the column temperature is 30-40 ℃; the flow rate is 0.8-1.0 ml/min; the sample volume is 50 mul;
the mass spectrum conditions are as follows: the Q-Q-TOF type tandem mass spectrometer is provided with an ESI ionization source and analysis data processing software; an ion source: an ESI ionization source; detecting in a positive ion mode; ion spray voltage 4500V; the temperature is 500 ℃; source gas 1: nitrogen pressure 50 psi; gas 2: nitrogen pressure 50 psi; gas curtain gas: nitrogen pressure 25 psi; TOF MS scan mode, declustering voltage (DP voltage): 80V; collision energy (CE voltage): 10 eV; swath scan pattern, declustering voltage (DP voltage): 80V; collision energy (CE voltage): 40eV, CES15 eV; the channels of the mass-to-charge ratio collection window performed by the Swath are 500-.
Further, selecting PEG specific fragment ions at m/z89.0611,133.0869,177.1102,221.1366,265.1622,309.1878,353.2108,397.2359; specific fragment ions of doxorubicin at m/z321.0838, 361.0785; the fragment ion at m/z 365.0735 specific for PEGylated doxorubicin.
And further, the method for measuring PEG and PEG adriamycin in the biological sample comprises the steps of sample pretreatment and standard curve preparation before measurement, liquid chromatography-quadrupole-time-of-flight mass spectrometry is carried out on the supernatant after pretreatment, a chromatogram is recorded, and the peak areas of PEG, adriamycin and PEG adriamycin are substituted into the standard curve to obtain the concentrations of PEG, adriamycin and PEG adriamycin.
The research firstly provides an LC-Q/Q/TOF tandem mass spectrum method of CID In-Quadrupole based on a continuous variable window acquisition technology, a TOF MS-Swath continuous variable window technology is established by utilizing the high resolution and high quality accuracy of a Quadrupole flight time mass spectrum (Triple TOF 5600), the collision energy corresponding to TOF MS is 10eV, and the collision energy corresponding to Swath is 40 eV; charged ions with different mass-to-charge ratio ranges sequentially enter a collision chamber Q2 by setting a continuously variable window, specific fragments are generated by energy collision of analytes, and the specific fragments are scanned and analyzed to establish a stable and reliable LC/MS/MS analysis method for PEG and PEG-modified drugs. More importantly, chromatographic mass spectrum peaks of PEG or PEG-modified drugs can be obtained in different mass-to-charge ratio windows through continuous change of the swath acquisition window, so that response ratios and distribution of the PEG or PEG-modified drugs in the different mass-to-charge ratio windows can be obtained according to peak areas. Meanwhile, due to the continuous sectional acquisition of the window, the ion transmission efficiency is high, the number of ions entering the collision chamber at the same time cannot exceed the threshold value of the cracking capacity of the collision chamber, the cracking efficiency is better, and the information abundance and the sensitivity of the obtained secondary fragments are better. In conclusion, by using the swing continuous variable window acquisition technology, primary parent ion information and secondary fragment information with high resolution can be simultaneously obtained by one-time acquisition, and qualitative and quantitative analysis of PEG and PEG drugs can be simultaneously completed. PEG-specific high-resolution specific fragment ions (at m/z89.0611133.0869,177.1102,221.1366,265.1622,309.1878,353.2108,397.2359, 2,3, 4, 5, 6, 7, 8 and 9 PEG units) and drug-specific ions (doxorubicin-specific fragment ions at m/z321.0838 and 361.0785; pegylated doxorubicin-specific fragment ions at m/z 365.0735), and the PEG-specific and drug-specific fragment ions are monitored to complete the analysis of PEG drugs and free PEG.
The invention has the advantages that:
1) the biological mass spectrometry method for qualitatively and quantitatively analyzing PEG and PEG drugs can be used for simultaneously obtaining high-resolution primary parent ion information and secondary fragment information through TOF MS Scan and Swath in sequence after one-time acquisition.
2) Distribution abundance of PEG and PEG drugs in different mass-to-charge ratio ranges can be determined through different acquisition channels of Swath, parent ions enter a collision chamber for cracking through the Swath in a segmented mode, transmission efficiency and cracking efficiency are higher, and sensitivity and selectivity are better.
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
Drawings
FIG. 1 is a flow chart of scanning of PEGylated drug-specific fragments by tandem mass spectrometry.
Fig. 2 is a relation of transmission efficiency to MS range.
FIG. 3 is a graph showing plasma concentration time profiles of doxorubicin and PEGylated doxorubicin after administration to the tail vein of the rat obtained in example 1.
FIG. 4 is a typical chromatogram for mPEG2000, doxorubicin and PEGylated doxorubicin obtained in example 1 (A, XIC of133.08+/-0.15 Da; B, XIC of 321.063+/-0.008 Da; C, XIC of 361.063+/-0.008 Da; D, XICof 365.057+/-0.008 Da).
FIG. 5 is a scanning mass spectrum of mPEG550 obtained in example 2.
FIG. 6 is a scanning mass spectrum of mPEG1000 obtained in example 2.
FIG. 7 is a mass to charge ratio distribution diagram of parent ions of mPEG550, mPEG750, mPEG2000 and mPEG5000 obtained in example 2.
FIG. 8 is a mass to charge ratio distribution diagram of mPEG 2K-doxorubicin parent ion obtained in example 2.
Detailed Description
Referring to fig. 1 and 2, the technical scheme of the invention is a biological mass spectrometry method for qualitatively and quantitatively analyzing PEG and pegylated drugs, which can acquire high-resolution primary parent ion information and secondary fragment information sequentially through TOF MS Scan and Swath at one time. The method uses liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-Q-TOFMS) for analysis and detection, separates the object to be detected containing PEG and PEG drugs by liquid chromatography, establishes a TOF MS-Swath continuously variable window, and respectively sets collision energy corresponding to the TOF MS and collision energy corresponding to the Swath; charged ions with different mass-to-charge ratio ranges sequentially enter the collision chamber Q2 by setting a continuously variable window, specific fragments are generated by energy collision of the object to be detected, and the object to be detected is detected by scanning and analyzing the specific fragments.
The invention can be used for detecting PEG and various PEGylated medicines in biological samples and non-biological samples. The method is very suitable for detecting PEG and PEG adriamycin in biological samples, and the detection steps are as follows:
the method comprises the following specific steps:
A. and (3) biological sample treatment:
1) 50 μ l of the biological sample was added to a polyethylene tube,
2) adding 50 mul of internal standard, mixing evenly by vortex,
3) adding 200 mul of acetonitrile, mixing evenly by vortex,
4) centrifuging at 12000rpm for 10 min, and transferring the supernatant to a polyethylene tube;
B. preparation of a standard curve:
1) PEG, adriamycin and PEG adriamycin stock solution are respectively diluted to 0.1, 0.2, 0.6, 1.0, 2.0, 6.0 and 10.0 mu g/mL by using acetonitrile-water (1/1, v/v) solution;
2) taking 50 μ l for liquid chromatography-tandem mass spectrometry, recording chromatogram, taking PEG, adriamycin and PEGylated adriamycin concentration as abscissa, taking PEG, adriamycin and PEGylated adriamycin peak area as ordinate, and using weighted W ═ 1/x2Performing regression operation by a least square method to obtain a linear regression equation, namely a standard curve;
C. measuring the content of free drugs and PEGylated drugs in the biological sample:
taking 50 mu l of the supernatant processed in the step A for liquid chromatography-tandem mass spectrometry, recording a chromatogram, substituting peak areas of PEG, adriamycin and PEG adriamycin into a standard curve, and obtaining the concentrations of PEG, adriamycin and PEG adriamycin; and C, preparing a standard curve in the step B and measuring the content of the adriamycin and the PEG adriamycin in the biological sample in the step C.
The chromatographic conditions are as follows: a high performance liquid chromatography system; a chromatographic column: 300SB C18 column, 150mm × 4.6mm I.D., particle size of 5 μm; mobile phase: water containing 0.1% formic acid by volume and acetonitrile containing 0.1% formic acid by volume, and carrying out gradient elution; the column temperature is 30-40 ℃; the flow rate is 0.8-1.0 ml/min; the sample volume is 50 mul;
the mass spectrum conditions are as follows: the Q-Q-Tof type tandem mass spectrometer is provided with an ESI ionization source and analysis data processing software; an ion source: an ESI ionization source; detecting in a positive ion mode; ion spray voltage 4500V; the temperature is 500 ℃; source gas 1: nitrogen pressure 50 psi; gas 2: nitrogen pressure 50 psi; gas curtain gas: nitrogen pressure 25 psi; TOF MS scan mode, declustering voltage (DP voltage): 80V; collision energy (CE voltage): 10 eV; swath scan pattern, declustering voltage (DP voltage): 80V; collision energy (CE voltage): 40eV, CES15 eV; the channels of the mass-to-charge ratio collection window performed by the swing are 500-;
in the PEG and PEG drug biological mass spectrometry analysis method, gradient elution in chromatographic conditions is performed according to the following procedures,
wherein A is water containing 0.1% by volume formic acid and B is acetonitrile containing 0.1% by volume formic acid.
The PEG micromolecule drug biological mass spectrum absolute quantitative method can also verify the method by using a quality control sample during the sample determination period; the quality control sample is prepared according to the following steps:
1) PEG, adriamycin and PEG adriamycin stock solution are respectively diluted to 0.2, 1.0 and 6.0 mu g/mL by using acetonitrile-water (1/1, v/v) solution;
2) three samples are taken for each concentration of PEG, adriamycin and PEG adriamycin, the concentration of the adriamycin and the PEG adriamycin is obtained according to a standard curve, and the accuracy of the quality control sample is calculated.
Two specific embodiments are described in detail below.
Example 1
PEG adriamycin with the drug loading equal to 1mg adriamycin is dissolved in 1mL of physiological saline, and is administrated to the tail vein of a rat, and the blood sampling time points are as follows: 0. 0.05, 0.083, 0.167, 0.25, 0.5, 1, 1.5, 2,3, 4, 6, 12, 24, 48, 72 h. The plasma levels of free doxorubicin and pegylated doxorubicin were determined after tail vein administration of the pegylated doxorubicin to rats and the plasma drug concentration time curve is shown in figure 3.
The method mainly comprises the following steps:
A. plasma sample pretreatment
1) 50 μ l of plasma sample was added to the polyethylene tube,
2) adding 50 mul of internal standard, mixing evenly by vortex,
3) adding 200 mul of acetonitrile, mixing evenly by vortex,
4) centrifuging at 12000rpm for 10 min, and transferring the supernatant to a polyethylene tube;
B. preparation of Standard Curve
1) PEG, adriamycin and PEG adriamycin stock solution are respectively diluted to 0.1, 0.2, 0.6, 1.0, 2.0, 6.0 and 10.0 mu g/mL by using acetonitrile-water (1/1, v/v) solution;
2) taking 50 μ l for liquid chromatography-tandem mass spectrometry, recording chromatogram, taking PEG, adriamycin and PEGylated adriamycin concentration as abscissa, taking PEG, adriamycin and PEGylated adriamycin peak area as ordinate, and using weighted W ═ 1/x2Performing regression operation by a least square method to obtain a linear regression equation, namely a standard curve;
as shown in table 1.
TABLE 1 typical standard curve for PEGylated doxorubicin biomembrane absolute quantitation method
C. Quality control sample preparation
The method is operated under the item of 'standard curve preparation', low, medium and high concentration (0.2, 1.0 and 6.0 mu g/mL) quality control samples are prepared, at least three samples are prepared for each concentration, and the concentration is obtained according to the standard curve.
The accuracy of the quality control samples was calculated, see table 2 specifically, and the method accuracy was investigated.
TABLE 2PEG doxorubicin biomolecular mass spectrometry absolute quantification method quality control sample accuracy
The conditions related to the PEG doxorubicin biological mass spectrum absolute quantitative method in the above steps are as follows:
the chromatographic conditions are as follows: a high performance liquid chromatography system; a chromatographic column: 300SB C18 column, 150mm × 4.6mm I.D., particle size of 5 μm; mobile phase: water containing 0.1% formic acid by volume and acetonitrile containing 0.1% formic acid by volume, and carrying out gradient elution; the column temperature is 40 ℃; the flow rate is 1 ml/min; the sample volume is 50 mul;
the gradient elution in the chromatographic conditions described, the procedure is shown in Table 3,
TABLE 3 gradient elution procedure
Wherein A is water containing 0.1% by volume formic acid and B is acetonitrile containing 0.1% by volume formic acid.
The mass spectrum conditions are as follows: the Q-Q-Tof type tandem mass spectrometer is provided with an ESI ionization source and analysis data processing software; an ion source: an ESI ionization source; detecting in a positive ion mode; ion spray voltage 4500V; the temperature is 500 ℃; source gas 1: nitrogen pressure 50 psi; gas 2: nitrogen pressure 50 psi; gas curtain gas: nitrogen pressure 25 psi; TOF MS scan mode, declustering voltage (DP voltage): 80V; collision energy (CE voltage): 10 eV; swath scan pattern, declustering voltage (DP voltage): 80V; collision energy (CE voltage): 40eV, CES15 eV; the channels of the mass-to-charge ratio collection window performed by the swing are 500-;
in view of the linearity of the typical standard curves of PEG2K, doxorubicin and PEGylated doxorubicin (Table 1) and the accuracy of the quality control samples (Table 2), the method for simultaneously determining the absolute quantification of PEG, PEGylated doxorubicin and free doxorubicin by using the biological mass spectrometry has the advantages of good linear relation, high accuracy, good reproducibility, sensitivity and reliability, and can be used for the absolute quantification of the PEG, PEGylated drugs and free drugs.
Example 2
PEG and PEG-modified adriamycin stock solutions are respectively diluted to 1.0 mu g/mL by using acetonitrile-water (1/1, v/v); taking 50 mu l of the mixture to carry out liquid chromatography-tandem mass spectrometry TOF MS-Swath analysis, recording a mass spectrogram and a chromatogram, and quantifying specific fragments of the medicines in channels with different mass-to-charge ratios to obtain the distribution of PEG and PEG-modified medicines in different mass-to-charge ratio ranges, wherein the mass-to-charge ratio range can be used for qualitative analysis of PEG and PEG-modified medicines with different molecular weights.
The chromatographic conditions are as follows: a high performance liquid chromatography system; a chromatographic column: 300SB C18 column, 150mm × 4.6mm I.D., particle size of 5 μm; mobile phase: water containing 0.1% formic acid by volume and acetonitrile containing 0.1% formic acid by volume, and carrying out gradient elution; the column temperature is 30 ℃; the flow rate is 0.8 ml/min; the sample volume is 50 mul;
the gradient elution in the chromatographic conditions described, the procedure is shown in Table 4,
TABLE 4 gradient elution procedure
Wherein A is water containing 0.1% by volume formic acid and B is acetonitrile containing 0.1% by volume formic acid.
The mass spectrum conditions are as follows: the Q-Q-Tof type tandem mass spectrometer is provided with an ESI ionization source and analysis data processing software; an ion source: an ESI ionization source; detecting in a positive ion mode; ion spray voltage 4500V; the temperature is 500 ℃; source gas 1: nitrogen pressure 50 psi; gas 2: nitrogen pressure 50 psi; gas curtain gas: nitrogen pressure 25 psi; TOF MS scan mode, declustering voltage (DP voltage): 80V; collision energy (CE voltage): 10 eV; swath scan pattern, declustering voltage (DP voltage): 80V; collision energy (CE voltage): 40eV, CES15 eV; the channels of the mass-to-charge ratio collection window performed by the Swath are 500-.
Claims (4)
1. An analysis and detection method of PEG and PEG drugs is characterized in that: analyzing and detecting by using a liquid chromatogram-quadrupole-time-of-flight mass spectrum, separating a substance to be detected containing PEG and PEG drugs by using the liquid chromatogram, establishing a TOF MS-Swath continuously variable window, and respectively setting collision energy corresponding to the TOF MS and collision energy corresponding to the Swath; charged ions with different mass-to-charge ratio ranges sequentially enter a collision chamber Q2 by setting a continuously variable window, specific fragments are generated by the object to be detected through energy collision, and the object to be detected is detected through scanning analysis of the specific fragments; the channels of the mass-to-charge ratio collection window performed by the swing are 500-; the selected PEG specific fragment ions are at m/z89.0611,133.0869,177.1102,221.1366,265.1622,309.1878,353.2108,397.2359, namely 2,3, 4, 5, 6, 7, 8 and 9 PEG units; the detection target in the object to be detected is PEG550, and the distribution window with the highest specific mass-to-charge ratio abundance is m/z 500-700; the detection target in the substance to be detected is PEG750, and the distribution window with the highest specific mass-to-charge ratio abundance is m/z 400-800; the detection target in the object to be detected is PEG2000, and the distribution window with the highest specific mass-to-charge ratio abundance is m/z 600-800; the detection target in the object to be detected is PEG5000, and the distribution window with the highest specific mass-to-charge ratio abundance is m/z 600-800; the detection target in the object to be detected is PEG2000-Dox, and the distribution window with the highest specific mass-to-charge ratio abundance is m/z 600-1000;
the chromatographic conditions are as follows: a high performance liquid chromatography system; a chromatographic column: 300SB C18 column, 150mm × 4.6mm I.D., particle size of 5 μm; mobile phase: water containing 0.1% formic acid by volume and acetonitrile containing 0.1% formic acid by volume, and carrying out gradient elution; the column temperature is 30-40 ℃; the flow rate is 0.8-1.0 ml/min; the sample volume is 50 ml;
the mass spectrum conditions are as follows: the Q-Q-TOF type tandem mass spectrometer is provided with an ESI ionization source and analysis data processing software; an ion source: an ESI ionization source; detecting in a positive ion mode; ion spray voltage 4500V; the temperature is 500 ℃; source gas 1: nitrogen pressure 50 psi; gas 2: nitrogen pressure 50 psi; gas curtain gas: nitrogen pressure 25 psi; TOF MS scan mode, declustering voltage: 80V; collision energy: 10 eV; swat scan pattern, declustering voltage: 80V; collision energy: 40eV, CES15 eV; the channels of the mass-to-charge ratio collection window performed by the Swath are 500-.
2. An analysis and detection method of PEG and PEG adriamycin is characterized in that: performing analysis and detection by using a liquid chromatogram-quadrupole-time-of-flight mass spectrometer, separating a substance to be detected containing PEG and PEG drugs by using the liquid chromatogram, establishing a TOFMS-Swath continuously variable window, and respectively setting collision energy corresponding to TOF MS and collision energy corresponding to Swath; charged ions with different mass-to-charge ratio ranges sequentially enter a collision chamber Q2 by setting a continuously variable window, a substance to be detected generates specific fragments through energy collision, the substance to be detected is detected through scanning and analyzing the specific fragments, and the detection conditions are as follows:
the chromatographic conditions are as follows: a high performance liquid chromatography system; a chromatographic column: 300SB C18 column, 150mm × 4.6mm I.D., particle size of 5 μm; mobile phase: water containing 0.1% formic acid by volume and acetonitrile containing 0.1% formic acid by volume, and carrying out gradient elution; the column temperature is 30-40 ℃; the flow rate is 0.8-1.0 ml/min; the sample volume is 50 ml;
the mass spectrum conditions are as follows: the Q-Q-TOF type tandem mass spectrometer is provided with an ESI ionization source and analysis data processing software; an ion source: an ESI ionization source; detecting in a positive ion mode; ion spray voltage 4500V; the temperature is 500 ℃; source gas 1: nitrogen pressure 50 psi; gas 2: nitrogen pressure 50 psi; gas curtain gas: nitrogen pressure 25 psi; TOF MS scan mode, declustering voltage: 80V; collision energy: 10 eV; swat scan pattern, declustering voltage: 80V; collision energy: 40eV, CES15 eV; the channels of the mass-to-charge ratio collection window performed by the Swath are 500-.
3. The method for analyzing and detecting PEG and PEG-ylated adriamycin according to claim 2, wherein: selecting PEG specific fragment ions at m/z89.0611,133.0869,177.1102,221.1366,265.1622,309.1878,353.2108,397.2359; specific fragment ions of doxorubicin at m/z321.0838, 361.0785; the fragment ion at m/z 365.0735 specific for PEGylated doxorubicin.
4. The method for analyzing and detecting PEG and PEG-ylated adriamycin according to claim 2, wherein: the method comprises the steps of sample pretreatment and standard curve preparation before the detection of PEG and PEG adriamycin in a biological sample, taking the supernatant after the pretreatment to perform liquid chromatography-quadrupole-time-of-flight mass spectrometry, recording a chromatogram, substituting the peak areas of the PEG, the adriamycin and the PEG adriamycin into the standard curve, and obtaining the concentrations of the PEG, the adriamycin and the PEG adriamycin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710706546.2A CN107589184B (en) | 2017-08-17 | 2017-08-17 | Analysis and detection method of PEG and PEG-modified drug and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710706546.2A CN107589184B (en) | 2017-08-17 | 2017-08-17 | Analysis and detection method of PEG and PEG-modified drug and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107589184A CN107589184A (en) | 2018-01-16 |
| CN107589184B true CN107589184B (en) | 2020-10-09 |
Family
ID=61042252
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710706546.2A Active CN107589184B (en) | 2017-08-17 | 2017-08-17 | Analysis and detection method of PEG and PEG-modified drug and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107589184B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109668977A (en) * | 2018-12-03 | 2019-04-23 | 大连理工大学 | The method for quantitatively determining of mPEG-PLA in a kind of biological sample |
| CN112285247B (en) * | 2020-12-30 | 2021-06-08 | 南京广祺医药科技有限公司 | Method for quantitatively determining PEGylated-IL-2(1A) in blood plasma and application |
| CN113419001A (en) * | 2021-06-17 | 2021-09-21 | 中荣凯特（北京）生物科技有限公司 | Method for quantitatively determining limaprost in biological sample |
| CN114002368A (en) * | 2021-12-30 | 2022-02-01 | 天津市食品安全检测技术研究院 | Method for determining illegal added components in health food by ultra-high performance liquid chromatography-quadrupole-time-of-flight high resolution mass spectrometry |
| CN114740116A (en) * | 2022-04-19 | 2022-07-12 | 中荣凯特（北京）生物科技有限公司 | Quantitative analysis method for multivalent PEGylated irinotecan prodrug and metabolite thereof in biological sample |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007044153A1 (en) * | 2005-08-31 | 2007-04-19 | Affymax, Inc. | Derivatization and low level detection of drugs in biological fluid and other solution matrices using a proxy marker |
| CN102507824A (en) * | 2011-11-01 | 2012-06-20 | 北京三元基因工程有限公司 | Analysis method for modification sites of polyethylene glycol modified protein |
| WO2014082733A1 (en) * | 2012-11-28 | 2014-06-05 | Eth Zurich | Method and tools for the determination of conformation and conformational changes of proteins and of derivatives thereof |
| CN103995042A (en) * | 2014-03-28 | 2014-08-20 | 深圳华大基因研究院 | Method for establishing protein sample SWATH ion library |
| CN104991016A (en) * | 2015-06-25 | 2015-10-21 | 吉林大学 | Quantitative assay method of PEG-modified medicine in biological sample |
-
2017
- 2017-08-17 CN CN201710706546.2A patent/CN107589184B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007044153A1 (en) * | 2005-08-31 | 2007-04-19 | Affymax, Inc. | Derivatization and low level detection of drugs in biological fluid and other solution matrices using a proxy marker |
| CN102507824A (en) * | 2011-11-01 | 2012-06-20 | 北京三元基因工程有限公司 | Analysis method for modification sites of polyethylene glycol modified protein |
| WO2014082733A1 (en) * | 2012-11-28 | 2014-06-05 | Eth Zurich | Method and tools for the determination of conformation and conformational changes of proteins and of derivatives thereof |
| CN103995042A (en) * | 2014-03-28 | 2014-08-20 | 深圳华大基因研究院 | Method for establishing protein sample SWATH ion library |
| CN104991016A (en) * | 2015-06-25 | 2015-10-21 | 吉林大学 | Quantitative assay method of PEG-modified medicine in biological sample |
Non-Patent Citations (3)
| Title |
|---|
| Applying ‘Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra’ (SWATH) for systematic toxicological analysis with liquid chromatography-high-resolution tandem mass spectrometry;Kathrin Arnhard et al;《Anal Bioanal Chem》;20141101;第407卷;405-414 * |
| Simultaneous quantitative analysis of polyethylene glycol (PEG), PEGylated paclitaxel and paclitaxel in rats by MSMSALL technique with hybrid quadrupole time-of-flight mass spectrometry;Heping Sun et al;《Journal of Pharmaceutical and Biomedical Analysis》;20170623;第145卷;255-261 * |
| SWATH Analysis of the Synaptic Proteome in Alzheimer Disease;Rachel Yoon Kyung Chang et al;《Neurochemistry International》;20150506;第87卷;1-12 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107589184A (en) | 2018-01-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107589184B (en) | Analysis and detection method of PEG and PEG-modified drug and application thereof | |
| AU2003218649B2 (en) | Mass spectrometry method for analysing mixtures of substances | |
| EP1530721B1 (en) | Method for characterizing biomolecules utilizing a result driven strategy | |
| Dunn | Mass spectrometry in systems biology: An introduction | |
| Korfmacher et al. | Demonstration of the capabilities of a parallel high performance liquid chromatography tandem mass spectrometry system for use in the analysis of drug discovery plasma samples | |
| Vogeser | Liquid chromatography-tandem mass spectrometry–application in the clinical laboratory | |
| CN110243985B (en) | Mass spectrum detection method of biomolecules | |
| CN112136043B (en) | Mass spectrometry method for detecting and quantifying liver function metabolites | |
| CN111089920A (en) | Liquid chromatography-mass spectrometry combined analysis and detection method for polysaccharide and application thereof | |
| WO2016196181A1 (en) | Mass spectrometry with quadrupole and ion mobility separation capabilities | |
| EP1952422A2 (en) | Mass spectrometer | |
| JP5065543B1 (en) | Method for detection and sequencing of post-translationally modified peptides | |
| CN111936851A (en) | Methods and systems for detecting vitamin D metabolites | |
| CN104991016A (en) | Quantitative assay method of PEG-modified medicine in biological sample | |
| King et al. | Collection of selected reaction monitoring and full scan data on a time scale suitable for target compound quantitative analysis by liquid chromatography/tandem mass spectrometry | |
| US20090256066A1 (en) | Method for calibrating detected mass in mass spectrometry | |
| Yang et al. | Determination of palonosetron in human plasma by ultra performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study | |
| CN104931637A (en) | Method for determining PEG content in biological sample | |
| Rivera et al. | Application of liquid chromatography-tandem mass spectrometry to the analysis of benzodiazepines in blood | |
| CN115808493A (en) | Method for detecting hormone in blood plasma by liquid chromatography-tandem mass spectrometry technology | |
| CN109085257B (en) | Method for simultaneously and quantitatively detecting astragaloside IV and cycloastragenol in mouse plasma | |
| Licea-Perez et al. | Development of an ultra-sensitive assay for the determination of an aminoalkyl glucosaminide 4-phosphate, GSK1795091, in plasma to support a first time in human study | |
| CN111413439A (en) | Method for determining metformin in blood plasma by rapid hydrophilic interaction chromatography-tandem mass spectrometry | |
| Zhang et al. | High-performance liquid chromatography–tandem mass spectrometry for the determination of pidotimod in human plasma and its application to a pharmacokinetic study | |
| Basov et al. | Investigation of the Chromatographic Behaviour of Metabolites from Blood Plasma by HPLC-MS/MS Using a Monolithic Column with a Sorbent Based on 1-Vinyl-1, 2, 4-triazole |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |