CN107589184A - The analyzing detecting method and its application of a kind of PEG and PEGylation medicine - Google Patents
The analyzing detecting method and its application of a kind of PEG and PEGylation medicine Download PDFInfo
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Abstract
The invention discloses a kind of PEG and the analyzing detecting method of PEGylation medicine, analysis detection is carried out using liquid chromatogram quadrupole rod flight time mass spectrum, determinand containing PEG and PEGylation medicine is separated by liquid chromatogram, and TOF MS Swath continuous variable windows are established, collision energy corresponding to collision energy corresponding to TOF MS and Swath is set respectively；Window by setting continuous variable makes the charged ion of different mass charge ratio ranges sequentially enter collision cell Q2, and determinand produces specific fragment through energy impact, and determinand is detected by being scanned analysis to specific fragment.The present invention establish it is a kind of can take into account simultaneously precursor scans and parent ion segmentation enter collision cell collide induction dissociation obtain particular fragment ion technical scheme, distribution of the high molecular polymer precursor ion in specific mass-to-charge ratio section can be obtained, completes the Qualitative and quantitative analysis of PEG and PEGylation medicine simultaneously according to parent ion and fragment ion.
Description
Technical field
The invention belongs to Pharmaceutical Analysis studying technological domain, is related to analyzing PEG and PEGylation medicine biological mass spectrometry method.
Background technology
In recent years, to adapt to the needs of medicament research and development, Pharmaceutical Analysis Science and Technology has obtained significant progress.In recent years
Come, the Liquid Chromatography-Tandem Mass Spectrometry technology (LC-MS/MS) developed rapidly provides possible solution party for PEGylation Pharmaceutical Analysis
Case.Compared with traditional immunology, HPLC, colorimetric method etc., Liquid Chromatography-Tandem Mass Spectrometry method is in the degree of accuracy, precision, selection
Property, each side such as sensitivity and quantitative dynamic range show greater advantage.At present, for the matter of PEGylation small-molecule drug
Spectrum quantitative analysis report is less, and its main cause is that the protein medicaments of PEGylation can take the method choice of proteomics
Feature peptide fragment after enzymolysis carries out quantitative analysis；But effective digestion means there is no to be hydrolyzed for PEGylation small-molecule drug
For free drug molecule.In addition, the range of molecular weight distributions of PEGylation medicine is wider, even if the PEG molecular weight of same polymeric degree
Also it is not unique, and the target chemical combination that the LC-MS/MS means based on scan patterns such as MRM can only determine to limited and molecular weight
Thing carries out quantitative analysis, and therefore, carrying out mass spectral analysis to the polymorphic macromolecule PEGylation medicine of multicomponent, there is very big to choose
War property.Cracking technique can provide abundant fragment ion information in first mass spectrometric in source.Using solving cluster voltage
(Declustering Potential, DP) influences ion and enters mass spectrographic speed, and the solution cluster voltage of taper hole is higher, ion velocity
Faster, losses of ions is smaller, and detection sensitivity is higher.Too high taper hole voltage can increase interionic collision, cause source implosion
Solution, produce fragment ion.These fragment ions can be not only used for the qualitative of material, can be used for MRM scan patterns while monitors
Parent ion and fragment ion realize quantitative analysis.By in ion gun add large energy solution cluster voltage (DP) make PEG and
Cracked in PEGylation medicine occurring source, generate specific fragment ion, select specific fragment ion to be monitored, completion pair
PEG and PEGylation medicine quantitative analysis；But because DP energy is smaller, CID in source collision efficiency has limited
System, this method are not appropriate for the analysis of the stronger PEGylation medicine of some binding abilities, are especially more difficult to get PEGylation medicine pin
To the particular fragment ion of medicine.When be bonded to small molecule (<When PEG molecular weight on 500) is larger (>When 10K), PEGylation
It is very close with PEGylation medicine chromatographic behavior, it is difficult to by chromatographic isolation, now simply by the monitoring specific fragments of PEG
To complete PEG and PEGylation medicine monitoring with regard to improper；Further, since DP collision energy is smaller, the sensitivity of this method
Also limited.MassallTechnology can provide PEG and PEGylation medicine quantitative approach to a certain extent, but due to
MassallTechnology is all charged particles is all broken into secondary fragment into collision cell by Q1, into collision cell from
Son, simultaneously because synchronization is excessive into collision cell ion populations, can significantly reduce the cracking of primary ion without selectivity
Efficiency and efficiency of transmission, sensitivity and selectivity can be restricted, simultaneously because PEG or PEGylation medicine can not be obtained not
With the chromatographic mass spectrometry peak response distribution situation in the range of mass-to-charge ratio, this method is applied to the qualitative analysis energy of PEG or PEGylation medicine
Power is limited.
The content of the invention
The technical problem to be solved in the present invention be establish one kind can take into account precursor scans simultaneously and parent ion be segmented into
Enter collision cell collide induction dissociation obtain particular fragment ion technical scheme, can obtain high molecular polymer precursor from
Son completes the qualitative and fixed of PEG and PEGylation medicine according to parent ion and fragment ion in the distribution in specific mass-to-charge ratio section simultaneously
Amount analysis.The purpose of the present invention is achieved through the following technical solutions：
A kind of PEG and PEGylation medicine analyzing detecting method, use liquid chromatogram-quadrupole time-of-flight mass spec-trometry (LC-
Q-Q-TOF MS) analysis detection is carried out, the determinand containing PEG and PEGylation medicine is separated by liquid chromatogram, and establishes
TOF MS-Swath continuous variable windows, collision energy corresponding to collision energy corresponding to TOF MS and Swath is set respectively；It is logical
Crossing the window of setting continuous variable makes the charged ion of different mass charge ratio ranges sequentially enter collision cell Q2, and determinand touches through energy
Hit and produce specific fragment, determinand is detected by being scanned analysis to specific fragment.
Further, collision energy corresponding to TOF MS is 10eV, and collision energy corresponding to Swath is 40eV.
Further, the passage of Swath progress mass-to-charge ratio acquisition window is followed successively by 500-600,600-700,700-800,
800-900,900-1000,1000-1100,1100-1200,1200-1250.
Further, the PEG specificity fragment ion of selection is at m/z 89.0611,133.0869,177.1102,
221.1366,265.1622,309.1878,353.2108,397.2359, i.e., 2,3,4,5,6,7,8,9
Individual PEG units.
Further, it is PEG550 target to be detected in determinand, and its specific mass-to-charge ratio abundance highest distribution window is m/z
500-700；It is PEG750 that target is detected in determinand, and its specific mass-to-charge ratio abundance highest distribution window is m/z 400-800；
It is PEG2000 that target is detected in determinand, and its specific mass-to-charge ratio abundance highest distribution window is m/z 600-800；Determinand
Middle detection target is PEG5000, and its specific mass-to-charge ratio abundance highest distribution window is m/z 600-800；Detected in determinand
Target is PEG2000-Dox, and its specific mass-to-charge ratio abundance highest distribution window is m/z 600-1000.
A kind of PEG and PEGylation adriamycin analyzing detecting method, use liquid chromatogram-quadrupole time-of-flight mass spec-trometry instrument
(LC-Q-Q-TOF MS) carries out analysis detection, and the determinand containing PEG and PEGylation medicine is separated by liquid chromatogram, and builds
Vertical TOF MS-Swath continuous variable windows, collision energy corresponding to collision energy corresponding to TOF MS and Swath is set respectively；
Window by setting continuous variable makes the charged ion of different mass charge ratio ranges sequentially enter collision cell Q2, and determinand is through energy
Collision produces specific fragment, determinand is detected by being scanned analysis to specific fragment, its testing conditions is：
Chromatographic condition is：Highly effective liquid phase chromatographic system；Chromatographic column：300SB C18 posts, 150mm × 4.6mm I.D., 5 μm
Particle diameter；Mobile phase：The water of 0.1% formic acid is accounted for containing percentage by volume and the acetonitrile of 0.1% formic acid is accounted for containing percentage by volume, and gradient is washed
It is de-；30~40 DEG C of column temperature；0.8~1.0ml/min of flow velocity；The μ l of sample size 50；
Mass Spectrometry Conditions are：Q-Q-TOF type tandem mass spectrometers, equipped with ESI ionization sources and Analyst data processing softwares；
Ion gun：ESI ionization sources；Positive ion mode detects；Ion injection electric 4500V；500 DEG C of temperature；Gas 1 in source：Nitrogen
Pressure 50psi；Gas 2：Nitrogen pressure 50psi；Curtain gas：Nitrogen pressure 25psi；TOF MS scan patterns, solve cluster voltage
(DP voltages)：80V；Collision energy (CE voltages)：10eV；Swath scan modes, solution cluster voltage (DP voltages)：80V；Impact energy
Measure (CE voltages)：40eV, CES 15eV；The passage that Swath carries out mass-to-charge ratio acquisition window is followed successively by 500-600,600-700,
700-800,800-900,900-1000,1000-1100,1100-1200,1200-1250.
Further, the PEG specificity fragment ion at m/z 89.0611,133.0869,177.1102 of selection,
221.1366,265.1622,309.1878,353.2108,397.2359；The specific fragment ion at m/z of adriamycin
321.0838,361.0785；The specific fragment ion at m/z 365.0735 of PEGylation adriamycin.
Further, determine PEG and PEGylation adriamycin in biological sample includes sample pre-treatments and standard before measurement
Curve preparation process, learn from else's experience and liquid chromatogram-quadrupole time-of-flight mass spec-trometry analysis, record are carried out by the supernatant after pre-treatment
Chromatogram, PEG, adriamycin and PEGylation adriamycin peak area are substituted into standard curve, try to achieve PEG, adriamycin and PEGylation Ah mould
Plain concentration.
This research proposes the LC-Q/Q/ of the CID In-Quadrupole based on continuous variable window acquisition technique first
TOF tandem mass spectrum methods, it is accurate using the high-resolution and high quality of level Four bar flight time mass spectrum (Triple TOF 5600)
Degree, establishes TOF MS-Swath continuous variable window techniques, collision energy corresponding to TOF MS is 10eV, is touched corresponding to Swath
It is 40eV to hit energy；Window by setting continuous variable makes the charged ion of different mass charge ratio ranges sequentially enter collision cell
Q2, analyte produce specific fragment through energy impact, by being scanned analysis to specific fragment, establish PEG and PEGylation
Drug substance stable, reliable LC/MS/MS analysis methods.Importantly, pass through swath acquisition window consecutive variations, Ke Yi
The chromatographic mass spectrometry peak of PEG or PEGylation medicine is obtained in different mass-to-charge ratio windows, so as to according to peak area it be drawn in difference
The response ratio of mass-to-charge ratio window and distribution.Simultaneously as the contiguous segmentation collection of window, ion transmission efficiency is high, with for the moment
The ion populations into collision cell are carved not over the threshold value of collision cell cracking ability, lysis efficiency is more preferable, and obtained two level is broken
The message abundance of piece and sensitivity are more preferable.To sum up, with swath continuous variable window acquisition techniques, it is same once to gather can
When obtain high-resolution one-level parent ion information and secondary fragment information, the qualitative of PEG and PEGylation medicine can be completed simultaneously
And quantitative analysis.The specific high-resolution particular fragment ions of PEG (at m/z89.0611133.0869,177.1102,
221.1366,265.1622,309.1878,353.2108,397.2359,2,3,4,5,6,7,8,9
PEG units) and drug-specific ion (the particular fragment ion at m/z 321.0838,361.0785 of adriamycin；PEGylation Ah
The particular fragment ion at m/z 365.0735 of mycin；), by monitor PEG specificity and drug specificity fragment ion come
Complete the analysis of PEGylation medicine and PEGylation, this method favorable reproducibility, high sensitivity, high-resolution fragment ion selection energy
The influence of interfering ion is enough effectively eliminated, is adapted to the analysis of PEG and PEGylation medicine in complex biological matrix.
Advantage of the invention is that：
1) once collection can pass sequentially through TOF MS Scan and Swath and meanwhile obtain high-resolution one-level mother from
The biological mass spectrometry method for being used for Qualitative and quantitative analysis PEG and PEGylation medicine of sub-information and secondary fragment information.
2) distribution of PEG and PEGylation medicine in different mass charge ratio ranges can be specified by Swath different acquisition passage
Abundance, parent ion enter collision cell by Swath segmentations and cracked, and efficiency of transmission and lysis efficiency are higher, sensitivity and selectivity
More preferably.
Below in conjunction with the accompanying drawings and embodiment is described in further detail to the present invention.
Brief description of the drawings
Fig. 1 is that tandem mass spectrum is scanned flow chart to PEGylation drug specificity fragment.
Fig. 2 is the relation of efficiency of transmission and MS range.
Fig. 3 is that the rat tail vein that embodiment 1 obtains gives adriamycin and PEGylation adriamycin blood medicine after PEGylation adriamycin
Cot curve.
Fig. 4 is mPEG2000, adriamycin and PEGylation adriamycin typical case chromatogram (A, the XIC of that embodiment 1 obtains
133.08+/-0.15Da；B,XIC of 321.063+/-0.008Da；C,XIC of 361.063+/-0.008Da；D,XIC
of 365.057+/-0.008Da)。
Fig. 5 is the mPEG550 scanning mass spectrograms that embodiment 2 obtains.
Fig. 6 is the mPEG1000 scanning mass spectrograms that embodiment 2 obtains.
Fig. 7 is the mPEG550 that embodiment 2 obtains, mPEG750, mPEG2000, and mPEG5000 m zs are distributed
Figure.
Fig. 8 is the mPEG2K- adriamycin m z distribution maps that embodiment 2 obtains.
Embodiment
Referring to Fig. 1,2, the technical scheme is that one kind, which can once gather can, passes sequentially through TOF MS Scan
Obtain high-resolution one-level parent ion information and secondary fragment information simultaneously with Swath is used for Qualitative and quantitative analysis PEG
And the biological mass spectrometry method of PEGylation medicine.The present invention uses liquid chromatogram-quadrupole time-of-flight mass spec-trometry (LC-Q-Q-TOF
MS analysis detection) is carried out, the determinand containing PEG and PEGylation medicine is separated by liquid chromatogram, and establishes TOF MS-
Swath continuous variable windows, collision energy corresponding to collision energy corresponding to TOF MS and Swath is set respectively；Pass through setting
The window of continuous variable makes the charged ion of different mass charge ratio ranges sequentially enter collision cell Q2, and determinand produces through energy impact
Specific fragment, determinand is detected by being scanned analysis to specific fragment.
PEG and a variety of PEGylation medicines that the present invention can be used in biological sample and non-biological specimen detection.It is especially suitable for
The detection of PEG and PEGylation adriamycin in biological sample, detection comprise the following steps that：
Comprise the following steps that：
A, biological sample is handled：
1) 50 μ l biological samples are added in polyethylene pipe,
2) 50 μ l internal standards are added, vortex mixes,
3) 200 μ l acetonitriles are added, vortex mixes,
4) 12000rpm is centrifuged 10 minutes, and supernatant is transferred to polyethylene pipe；
B, prepared by standard curve：
1) PEG, adriamycin and PEGylation adriamycin storing solution are diluted to respectively using acetonitrile-water (1/1, v/v) solution
0.1、0.2、0.6、1.0、2.0、6.0、10.0μg/mL；
2) take 50 μ l to carry out liquid chromatography-tandem mass spectrometry analysis, chromatogram is recorded, with PEG, adriamycin and PEGylation Ah mould
Plain concentration is abscissa, and PEG, adriamycin and PEGylation adriamycin peak area are ordinate, with weighting W=1/x2Least square method
Carry out regressing calculation, the as linear regression equation tried to achieve, standard curve；
C, free drug and PEGylation medicine assay in biological sample：
The μ l of the supernatant after being handled by step A 50 that learn from else's experience carry out liquid chromatography-tandem mass spectrometry analysis, record chromatogram, will
PEG, adriamycin and PEGylation adriamycin peak area substitute into standard curve, try to achieve PEG, adriamycin and PEGylation doxorubicin concentration；Institute
Adriamycin and PEGylation doxorubicin content measure in the step B standard curve preparation stated and step C biological sample.
Chromatographic condition is：Highly effective liquid phase chromatographic system；Chromatographic column：300SB C18 posts, 150mm × 4.6mm I.D., 5 μm
Particle diameter；Mobile phase：The water of 0.1% formic acid is accounted for containing percentage by volume and the acetonitrile of 0.1% formic acid is accounted for containing percentage by volume, and gradient is washed
It is de-；30~40 DEG C of column temperature；0.8~1.0ml/min of flow velocity；The μ l of sample size 50；
Mass Spectrometry Conditions are：Q-Q-Tof type tandem mass spectrometers, equipped with ESI ionization sources and Analyst data processing softwares；
Ion gun：ESI ionization sources；Positive ion mode detects；Ion injection electric 4500V；500 DEG C of temperature；Gas 1 in source：Nitrogen
Pressure 50psi；Gas 2：Nitrogen pressure 50psi；Curtain gas：Nitrogen pressure 25psi；TOF MS scan patterns, solve cluster voltage
(DP voltages)：80V；Collision energy (CE voltages)：10eV；Swath scan modes, solution cluster voltage (DP voltages)：80V；Impact energy
Measure (CE voltages)：40eV,CES15eV；The passage that Swath carries out mass-to-charge ratio acquisition window is followed successively by 500-600,600-700,
700-800,800-900,900-1000,1000-1100,1100-1200,1200-1250；
In the PEG and PEGylation medicine biological mass spectrometry analysis method of the present invention, the gradient elution in described chromatographic condition,
Program see the table below,
Wherein A is the water that 0.1% formic acid is accounted for containing percentage by volume, and B is the acetonitrile that 0.1% formic acid is accounted for containing percentage by volume.
The PEGylation small-molecule drug biological mass spectrometry absolute quantification method of the present invention, it can also be utilized during sample determines
Quality control samples are verified to method；Described Quality control samples, are prepared in accordance with the following steps：
1) PEG, adriamycin and PEGylation adriamycin storing solution are diluted to respectively using acetonitrile-water (1/1, v/v) solution
0.2、1.0、6.0μg/mL；
2) PEG, adriamycin and PEGylation adriamycin take three samples per concentration, according to standard curve, draw adriamycin and
PEGylation doxorubicin concentration, calculate the Quality control samples degree of accuracy.
Described in detail below by two specific embodiments.
Embodiment 1
Drugloading rate is equal to the PEGylation adriamycin 1mL physiological saline solutions of 1mg adriamycins, rat tail vein is administered,
Blood sampling time point is as follows：0、0.05、0.083、0.167、0.25、0.5、1、1.5、2、3、4、6、12、24、48、72h.Measure is big
Caudal vein gives the content of dissociate in blood plasma after PEGylation adriamycin adriamycin and PEGylation adriamycin, during plasma drug level
Half interval contour is shown in Fig. 3.
Key step is as follows：
A, plasma sample pre-processes
1) 50 μ l plasma samples are added in polyethylene pipe,
2) 50 μ l internal standards are added, vortex mixes,
3) 200 μ l acetonitriles are added, vortex mixes,
4) 12000rpm is centrifuged 10 minutes, and supernatant is transferred to polyethylene pipe；
B, prepared by standard curve
1) PEG, adriamycin and PEGylation adriamycin storing solution are diluted to respectively using acetonitrile-water (1/1, v/v) solution
0.1、0.2、0.6、1.0、2.0、6.0、10.0μg/mL；
2) take 50 μ l to carry out liquid chromatography-tandem mass spectrometry analysis, chromatogram is recorded, with PEG, adriamycin and PEGylation Ah mould
Plain concentration is abscissa, and PEG, adriamycin and PEGylation adriamycin peak area are ordinate, with weighting W=1/x2Least square method
Carry out regressing calculation, the as linear regression equation tried to achieve, standard curve；
As shown in table 1.
The PEGylation adriamycin biological mass spectrometry absolute quantification method representative standard curve of table 1
C, prepared by Quality control samples
By being operated under " standard curve preparation " item, basic, normal, high three concentration (0.2,1.0,6.0 μ g/mL) quality control is prepared
Sample preparation product, per concentration at least three samples, according to standard curve, draw concentration.
The Quality control samples degree of accuracy is calculated, is specifically shown in Table 2, investigates method accuracy.
The PEGylation adriamycin biological mass spectrometry absolute quantification method Quality control samples degree of accuracy of table 2
The condition for being related to PEGylation adriamycin biological mass spectrometry absolute quantification method measure in above-mentioned steps is as follows：
Chromatographic condition is：Efficient liquid phase liquid chromatographic system；Chromatographic column：300SB C18 posts, 150mm × 4.6mm I.D.,
5 μm of particle diameters；Mobile phase：The water of 0.1% formic acid is accounted for containing percentage by volume and the acetonitrile of 0.1% formic acid is accounted for containing percentage by volume, ladder
Degree elution；40 DEG C of column temperature；Flow velocity 1ml/min；The μ l of sample size 50；
Gradient elution in described chromatographic condition, program are shown in Table 3,
The gradient elution program of table 3
Wherein A is the water that 0.1% formic acid is accounted for containing percentage by volume, and B is the acetonitrile that 0.1% formic acid is accounted for containing percentage by volume.
Mass Spectrometry Conditions are：Q-Q-Tof type tandem mass spectrometers, equipped with ESI ionization sources and Analyst data processing softwares；
Ion gun：ESI ionization sources；Positive ion mode detects；Ion injection electric 4500V；500 DEG C of temperature；Gas 1 in source：Nitrogen
Pressure 50psi；Gas 2：Nitrogen pressure 50psi；Curtain gas：Nitrogen pressure 25psi；TOF MS scan patterns, solve cluster voltage
(DP voltages)：80V；Collision energy (CE voltages)：10eV；Swath scan modes, solution cluster voltage (DP voltages)：80V；Impact energy
Measure (CE voltages)：40eV,CES15eV；The passage that Swath carries out mass-to-charge ratio acquisition window is followed successively by 500-600,600-700,
700-800,800-900,900-1000,1000-1100,1100-1200,1200-1250；
In view of linear (table 1) and Quality control samples of PEG2K, adriamycin and PEGylation adriamycin representative standard curve
The degree of accuracy (table 2), it is of the present invention it is a kind of determine PEG simultaneously, the biological mass spectrometry of PEGylation adriamycin and free adriamycin is exhausted
It is good to quantitative approach linear relationship, the degree of accuracy is high, favorable reproducibility and sensitive, reliable, available for above-mentioned PEG, PEGylation medicine
And the absolute quantitation of free drug.
Embodiment 2
PEG and PEGylation adriamycin storing solution are diluted to 1.0 μ g/mL respectively using acetonitrile-water (1/1, v/v) solution；Take
50 μ l carry out liquid chromatography-tandem mass spectrometry TOF MS-Swath analyses, mass spectrogram and chromatogram are recorded, to different mass-to-charge ratio passages
The specific fragment of interior medicine is quantified, and is drawn the distribution of PEG and PEGylation medicine in different mass charge ratio ranges, be can be used for
The PEG and PEGylation medicine of different molecular weight qualitative analysis.
Chromatographic condition is：Efficient liquid phase liquid chromatographic system；Chromatographic column：300SB C18 posts, 150mm × 4.6mm I.D.,
5 μm of particle diameters；Mobile phase：The water of 0.1% formic acid is accounted for containing percentage by volume and the acetonitrile of 0.1% formic acid is accounted for containing percentage by volume, ladder
Degree elution；30 DEG C of column temperature；Flow velocity 0.8ml/min；The μ l of sample size 50；
Gradient elution in described chromatographic condition, program are shown in Table 4,
The gradient elution program of table 4
Wherein A is the water that 0.1% formic acid is accounted for containing percentage by volume, and B is the acetonitrile that 0.1% formic acid is accounted for containing percentage by volume.
Mass Spectrometry Conditions are：Q-Q-Tof type tandem mass spectrometers, equipped with ESI ionization sources and Analyst data processing softwares；
Ion gun：ESI ionization sources；Positive ion mode detects；Ion injection electric 4500V；500 DEG C of temperature；Gas 1 in source：Nitrogen
Pressure 50psi；Gas 2：Nitrogen pressure 50psi；Curtain gas：Nitrogen pressure 25psi；TOF MS scan patterns, solve cluster voltage
(DP voltages)：80V；Collision energy (CE voltages)：10eV；Swath scan modes, solution cluster voltage (DP voltages)：80V；Impact energy
Measure (CE voltages)：40eV,CES15eV；The passage that Swath carries out mass-to-charge ratio acquisition window is followed successively by 500-600,600-700,
700-800,800-900,900-1000,1000-1100,1100-1200,1200-1250.
Claims (9)
1. a kind of PEG and PEGylation medicine analyzing detecting method, it is characterised in that：During using liquid chromatogram-quadrupole rod-flight
Between mass spectrum carry out analysis detection, the determinand containing PEG and PEGylation medicine is separated by liquid chromatogram, and establish TOF MS-
Swath continuous variable windows, collision energy corresponding to collision energy corresponding to TOF MS and Swath is set respectively；Pass through setting
The window of continuous variable makes the charged ion of different mass charge ratio ranges sequentially enter collision cell Q2, and determinand produces through energy impact
Specific fragment, determinand is detected by being scanned analysis to specific fragment.
2. PEG according to claim 1 and PEGylation medicine analyzing detecting method, it is characterised in that：Corresponding to TOF MS
Collision energy is 10eV, and collision energy corresponding to Swath is 40eV.
3. PEG according to claim 1 and PEGylation medicine analyzing detecting method, it is characterised in that：Swath carries out matter
Lotus is followed successively by 500-600,600-700,700-800,800-900,900-1000,1000-1100 than the passage of acquisition window,
1100-1200,1200-1250.
4. PEG according to claim 1 and PEGylation medicine analyzing detecting method, it is characterised in that：The PEG of selection is special
Different in nature fragment ion is at m/z 89.0611,133.0869,177.1102,221.1366,265.1622,
309.1878,353.2108,397.2359, i.e., 2,3,4,5,6,7,8,9 PEG units.
5. PEG according to claim 1 and PEGylation medicine analyzing detecting method, it is characterised in that：Detected in determinand
Target is PEG550, and its specific mass-to-charge ratio abundance highest distribution window is m/z 500-700；Target is detected in determinand is
PEG750, its specific mass-to-charge ratio abundance highest distribution window are m/z 400-800；It is PEG2000 that target is detected in determinand,
Its specific mass-to-charge ratio abundance highest distribution window is m/z 600-800；It is PEG5000 that target is detected in determinand, and its is specific
Mass-to-charge ratio abundance highest distribution window is m/z 600-800；It is PEG2000-Dox that target is detected in determinand, its specific matter
Lotus is m/z 600-1000 than abundance highest distribution window.
6. the analyzing detecting method of PEG described in claim 1 and PEGylation medicine is applied to PEG and PEGylation medicine in biological sample
Detection.
7. a kind of PEG and PEGylation adriamycin analyzing detecting method, it is characterised in that：Use liquid chromatogram-quadrupole rod-flight
Time mass spectrum instrument carries out analysis detection, the determinand containing PEG and PEGylation medicine is separated by liquid chromatogram, and establish TOF
MS-Swath continuous variable windows, collision energy corresponding to collision energy corresponding to TOF MS and Swath is set respectively；Pass through
The window of setting continuous variable makes the charged ion of different mass charge ratio ranges sequentially enter collision cell Q2, and determinand is through energy impact
Specific fragment is produced, determinand is detected by being scanned analysis to specific fragment, its testing conditions is：
Chromatographic condition is：Highly effective liquid phase chromatographic system；Chromatographic column：300SB C18 posts, the mm I.D. of 150 mm × 4.6,5 μm of grains
Footpath；Mobile phase：The water of 0.1% formic acid is accounted for containing percentage by volume and the acetonitrile of 0.1% formic acid, gradient elution are accounted for containing percentage by volume；
30 ~ 40 DEG C of column temperature；The ml/min of flow velocity 0.8 ~ 1.0；Sample size 50ml；
Mass Spectrometry Conditions are：Q-Q-TOF type tandem mass spectrometers, equipped with ESI ionization sources and Analyst data processing softwares；Ion
Source：ESI ionization sources；Positive ion mode detects；The V of ion injection electric 4500；500 DEG C of temperature；Gas 1 in source：Nitrogen pressure
50 psi；Gas 2：The psi of nitrogen pressure 50；Curtain gas：The psi of nitrogen pressure 25；TOF MS scan patterns, solve cluster voltage：
80 V；Collision energy：10 eV；Swath scan modes, solve cluster voltage：80 V；Collision energy：The eV of 40 eV, CES 15；
The passage that Swath carries out mass-to-charge ratio acquisition window is followed successively by 500-600,600-700,700-800,800-900,900-1000,
1000-1100,1100-1200,1200-1250.
8. PEG according to claim 7 and PEGylation adriamycin analyzing detecting method, it is characterised in that：The PEG of selection
Specific fragment ion at m/z 89.0611,133.0869,177.1102,221.1366,265.1622,
309.1878, 353.2108, 397.2359；The specific fragment ion at m/z 321.0838,361.0785 of adriamycin；
The specific fragment ion at m/z 365.0735 of PEGylation adriamycin.
9. PEG according to claim 7 and PEGylation adriamycin analyzing detecting method, it is characterised in that：Determine biological sample
PEG and PEGylation adriamycin include sample pre-treatments and standard curve preparation process before measurement in product, learn from else's experience by pre-treatment
Supernatant afterwards carries out liquid chromatogram-quadrupole time-of-flight mass spec-trometry analysis, chromatogram is recorded, by PEG, adriamycin and PEGylation
Adriamycin peak area substitutes into standard curve, tries to achieve PEG, adriamycin and PEGylation doxorubicin concentration.
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