CN114740116A - Quantitative analysis method for multivalent PEGylated irinotecan prodrug and metabolite thereof in biological sample - Google Patents

Quantitative analysis method for multivalent PEGylated irinotecan prodrug and metabolite thereof in biological sample Download PDF

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CN114740116A
CN114740116A CN202210408609.7A CN202210408609A CN114740116A CN 114740116 A CN114740116 A CN 114740116A CN 202210408609 A CN202210408609 A CN 202210408609A CN 114740116 A CN114740116 A CN 114740116A
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peg
cpt
prodrug
irinotecan
metabolites
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顾景凯
宋诗文
孙冬
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Zhongrong Kaite Beijing Biotechnology Co ltd
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Abstract

The invention discloses a quantitative analysis method for a polyvalent PEG irinotecan prodrug and metabolites thereof in a biological sample. Measuring by high performance liquid chromatography-time of flight mass spectrometry by combining a full spectrum scanning mode of biological mass spectrometry with a scanning mode of daughter ions; grouping and separating the n-valent PEGylated irinotecan prodrug and the mixture of metabolites with different forms in vivo in a biological sample by high performance liquid chromatography; and (4) selecting common characteristic fragments through mass spectrum, and quantifying each component. The method provided by the invention is suitable for clinical prodrug pharmacokinetics research and detection of the n-valent PEG prodrug in the complex biological matrix, and has good selectivity and high separation degree.

Description

Quantitative analysis method for multivalent PEGylated irinotecan prodrug and metabolite thereof in biological sample
Technical Field
The invention belongs to the technical field of drug analysis research, and particularly relates to a method for synchronously and quantitatively analyzing a biological mass spectrum of n-valent PEG (polyethylene glycol) irinotecan prodrug and drug-related metabolites of different forms in a biological sample.
Background
Irinotecan (CPT-11) is a semisynthetic camptothecin derivative, belonging to the class of prodrugs, whose antitumor activity derives from its active metabolite SN38, after its conversion by carboxylesterase. CPT-11 inhibits tumor cell proliferation and induces apoptosis by inhibiting topoisomerase I (Topo I), is a broad-spectrum cytotoxic antitumor drug, and is mainly used for first-line and second-line treatment of colorectal cancer and other solid tumors in clinic at present. However, irinotecan has poor water solubility, short half-life and serious toxic and side effects, which limits the clinical application of irinotecan. Irinotecan is structurally modified by polyethylene glycol (PEG), so that the bioavailability of the medicine can be further improved, and the toxic and side effects are reduced.
PEG is a non-toxic, harmless and biocompatible biomedical polymer material certified by the Food and Drug Administration (FDA), and is often used as a hydrophilic end to modify drugs and nano-formulations. Pegylation is a technique by which a polyethylene glycol polymer is covalently attached to a therapeutic drug, and polyethylene glycol-modified drug molecules have the following advantages: 1. increase the water solubility of the drug; 2. the toxicity is reduced; 3. the half-life period of the medicine circulation is prolonged, the times of medicine taking are reduced, the compliance of patients is improved, the life quality is improved, and the treatment cost is reduced; 4. reducing enzyme degradation and improving bioavailability.
Compared with single straight-chain PEG, the n-valent PEG micromolecule drug can greatly improve the drug loading rate. However, the in vivo dynamic drug release profile and pharmacokinetic profile are much more complex than that of single linear PEGylated drugs. The bottleneck problems of the current research are as follows: for single linear PEGylation drugs, in vivo pharmacokinetics studies have been mainly performed around PEGylation drugs, PEG and free drugs. The n-valent PEGylated small molecule drug is required to simultaneously examine the in vivo change rule of PEGylated drugs with different valence states besides the above parts. The in vivo fate of the linear chain n-valent PEG micromolecule drug is explained, and the method has very important pharmacological and toxicological significance.
The molecular weight of PEG is not unique at present and the polydispersity is highThe conventional mass spectrum quantitative analysis methods of the seed polymer are a Collision-induced dissociation (CID) strategy and a full spectrum scanning (MS) strategyALL). These two strategies differ from: CID is primarily dependent on declustering voltage (DP) within the ion source, which generally provides lower energy, resulting in inefficient fragmentation and generally relatively lower sensitivity. MS (Mass Spectrometry)ALLMainly depends on the Collision Energy (CE) in the collision chamber, has higher energy and thus higher cracking efficiency, and is an ideal method for quantitatively analyzing the polymer material. But using MSALLThe quantification of the technology is performed on the premise that all compounds with the same fragment are chromatographically separated. Generally speaking, the influence of the quantity of the small-molecule drug loaded on the PEG on the polarity is very little, namely the difference of the chromatographic retention behaviors is small, and the PEG- (CPT-11) is realizednWith PEG- (CPT-11)n(m)There are currently significant challenges to chromatographic separation. In realizing the prodrug PEG- (CPT-11)nAnd prodrug metabolite PEG- (CPT-11) of different drug loading statesn(m)The separated irinotecan (CPT-11), the irinotecan active metabolite (SN38) and the PEG non-drug part are grouped and synchronously quantified while the chromatogram is separated, so that the work is difficult to add.
Disclosure of Invention
In order to overcome the technical problems, the invention aims to establish an analysis method which has good selectivity and high separation degree and can simultaneously and quantitatively determine n-valent PEGylated irinotecan prodrug and metabolites thereof with different forms in vivo in a biological sample.
In order to achieve the purpose, the technical scheme of the invention is as follows: chromatographic conditions were established to PEGylate irinotecan prodrug PEG- (CPT-11)nAnd metabolites with different forms in vivo realize chromatographic separation; and find PEG-modified irinotecan prodrug PEG- (CPT-11)nAnd prodrug metabolite PEG- (CPT-11) of different drug loading statesn(m)A characteristic fragment common to each component in (a).
The measurement is carried out by adopting high performance liquid chromatography-time-of-flight mass spectrometry, and a biological mass spectrometry full spectrum scanning Mode (MS)ALL) Binding to Product Ion scan pattern (Product Ion). The invention realizes raw material generation by using chromatographyPEG (polyethylene glycol) prodrug of PEGylated irinotecan in different valence states in sample- (CPT-11)n(wherein n is 2-12), PEG (PEGylated irinotecan prodrug metabolite PEG- (CPT-11)n(m)(wherein m is 1-11), PEG non-drug part, irinotecan (CPT-11), irinotecan active metabolite (SN38) and endogenous interference, and synchronously and quantitatively analyzing polymorphic components of the irinotecan. For the PEG non-drug moiety, PEG-linker arms and PEG-moiety linker arms were alternatively quantitated using PEG20K standard.
The specific scheme is as follows:
a quantitative analysis method for polyvalent PEGylated irinotecan prodrug and metabolites thereof in a biological sample adopts high performance liquid chromatography-time-of-flight mass spectrometry to carry out determination, and adopts a biological mass spectrometry whole spectrum scanning Mode (MS)ALL) Binding Ion scan mode (Product Ion); firstly, grouping and separating the n-valent PEG irinotecan prodrug and the mixture of metabolites with different forms in vivo in a biological sample by high performance liquid chromatography to ensure that the isomers or the homologous derivatives in the same group have the same liquid phase retention behavior; then, common characteristic fragments are selected through mass spectrum, so that absolute quantification of each component and substitution quantification of PEG non-drug parts are realized;
the n-valent PEGylated irinotecan prodrug is expressed as PEG- (CPT-11)n(ii) a Wherein n represents a valence state, and means a PEG- (CPT-11)nThe maximum value of the parent drug is loaded on the prodrug molecule, and n is 2-12;
the mixture of metabolites of different morphologies in vivo of n-valent pegylated irinotecan prodrug comprises: PEGylated irinotecan prodrug metabolite PEG- (CPT-11) of different drug loading statesn(m)The components, dissociated irinotecan CPT-11, irinotecan active metabolite SN38 and PEG non-drug part; irinotecan metabolites including both active and inactive metabolites;
wherein, PEG- (CPT-11)n(m)In the formula, m is the number of the residual CPT-11 residues on PEG, m is 1-11, and the prodrug metabolites with the same m value are isomers;
wherein, the series quadrupole time-of-flight mass spectrum is selected from PEG- (CPT-11)nAnd PEG- (CPT-11)n(m)Common characteristic bitsPiece m/z 569.27, fragment m/z 587.28 characteristic of CPT-11, fragment m/z 393.15 characteristic of cleavage reaction of SN38 → 349.14, fragment m/z 177.11 characteristic of PEG non-drug moiety grouping.
Wherein, PEG non-drug part refers to prodrug metabolite containing PEG without connecting with parent drug and parent drug metabolite, such as PEG, PEG-linking arm and PEG-part linking arm, which are homologous derivatives;
wherein, PEG comprises straight chain, branched chain, single end capping and block PEG;
the connection mode of parent drugs in n-valent PEG chemical drugs comprises direct connection with PEG through ester bonds and connection through connecting arms, and the connection sites of the parent drugs are 4-hydroxy.
The specific mass spectrometry flow is as follows: 1) MS (Mass Spectrometry)ALLIn the scanning mode, all the objects pass through the Q1, the objects are broken up through proper Collision Energy (CE) in the Q2 process, and the TOF mass analyzer scans all passing ions to obtain high-resolution information of all fragments. Under the passage of CE50, PEG- (CPT-11) was extracted from the obtained fragmentsnWith PEG- (CPT-11)n(m)Common characteristic fragment 569.27 + -0.02 Da, characteristic fragment 177.11 + -0.02 Da of PEG non-drug part extracted under CE30 channel, and characteristic fragment 587.28 + -0.02 Da of irinotecan extracted. 2) In Product Ion scan mode, CE is set to 40 and SN38 quantifies Ion pairs with m/z of 393.15 → 349.14.
Further, preferred liquid chromatography conditions are: a chromatographic column: agilent 5TC-C18(2) column, 250mm × 4.6mm, 5 μm particle size; mobile phase: water phase: aqueous, organic phase containing 0.1% formic acid: 50% acetonitrile + 50% methanol (containing 0.1% formic acid); gradient elution; the column temperature is 30-60 ℃ (preferably 50 ℃); flow rate: 0.5-2.0 mL/min (preferably 1.0 mL/min); the sample amount is 5-20 μ L (preferably 10-20 μ L).
Further, the gradient elution procedure in chromatographic conditions started at an organic phase ratio of 10% and subsequently transited to 70% with a maximum organic phase ratio of 95%. The rate of increase of the organic phase proportion from 10% to 70% was 30%/min, and the rate of increase of the organic phase proportion from 70% to 95% was 1.92%/min.
Preferred mass spectrometry conditions are: triple TOFTM6600+A tandem mass spectrometer of the type equipped with an ESI ionization source; detecting in a positive ion mode; ion spray voltage 5000-; temperature 450-550 deg.C (preferably 550 deg.C); source gas 1: nitrogen pressure 40 + -10 psi (preferably 50 psi); gas 2: nitrogen pressure 40 + -10 psi (preferably 50 psi); gas curtain gas: nitrogen pressure 40-55psi (preferably 40 psi); cluster-splitting voltage: 50V; MS (Mass Spectrometry)ALLThe scanning range of the scanning mode is 50-2500 Da; the scanning range of the Product Ion scanning mode is 200-400 Da; the collision energy is 10-50eV, wherein PEG- (CPT-11)nWith PEG- (CPT-11)n(m)The channel collision energy was 50eV, the PEG non-drug moiety and irinotecan channel collision energy was 30eV, and the SN38 channel collision energy was 40 eV.
Further, before the measurement, the biological sample is subjected to pretreatment and a preparation step of a standard curve; the supernatant after pretreatment is subjected to high performance liquid chromatography-time-of-flight mass spectrometry, and full spectrum scanning (MS)ALL) Binding partner Ion scan (Product Ion) analysis, and chromatogram were recorded. Substituting peak areas of n-valent PEG irinotecan prodrug and various components of metabolites with different forms in vivo into a standard curve to obtain corresponding concentrations.
Pretreatment of biological samples adopts a method of precipitating protein to extract n-valent PEG irinotecan prodrug PEG- (CPT-11)nAnd metabolites with different forms in the body, and the pretreatment methods of different kinds of complex biological samples are slightly different, wherein the biological samples include but are not limited to blood plasma, urine and feces. When the biological sample is plasma, the pretreatment is as follows: treating the obtained blood plasma with anticoagulant and antagonist, precipitating protein, centrifuging, and taking supernatant for dilution; when the biological sample is urine or bile, the pretreatment is as follows: precipitating protein, centrifuging, and taking supernatant for dilution; when the biological sample is feces, the pretreatment is as follows: freeze drying, crushing, re-dissolving, vortex centrifuging, taking supernatant, precipitating protein, centrifuging and diluting the supernatant.
The quantitative analysis method can be used for quantitative detection of the preclinical n-valent PEGylated irinotecan prodrug, such as plasma pharmacokinetics, tissue distribution, metabolism and excretion measurement.
The quantitative analysis method of the invention is used for PEG- (CPT-11) of the PEGylated irinotecan prodrug in rat blood matrix3The detection range of (2) is 3-500 mug/mL, and the lower limit mass number of the quantification is 3 mug/mL; PEG- (CPT-11) which is one of metabolites thereof3(2)The detection range of (2) is 5-250 mug/mL, and the lower limit mass number of the quantification is 5 mug/mL; PEG- (CPT-11) which is one of its metabolites3(1)The detection range of (1-50) mu g/mL, and the lower limit mass number of the quantification is 1 mu g/mL; the detection range of the PEG non-drug part of one metabolite is 1-50 mug/mL, and the lower limit mass number of the quantification is 1 mug/mL; the detection range of CPT-11 of one of the metabolites is 20-1000ng/mL, and the lower limit mass number of the quantification is 20 ng/mL; the detection range of SN38 of one of its metabolites was 10-500ng/mL, and the lower limit mass number of the quantitation was 10 ng/mL.
PEG- (CPT-11) for PEGylated irinotecan prodrug in rat urine matrix3The detection range of (1) is 1-2000 mug/mL, and the lower limit mass number of the quantification is 1 mug/mL; PEG- (CPT-11) which is one of its metabolites3(2)The detection range of (2) is 3-900 mug/mL, and the lower limit mass number of the quantification is 3 mug/mL; PEG- (CPT-11) which is one of metabolites thereof3(1)The detection range of (1-300) is 1-300 mug/mL, and the lower limit mass number of the quantification is 1 mug/mL; the detection range of the PEG non-drug part of one metabolite is 1-300 mug/mL, and the lower limit mass number of the quantification is 1 mug/mL; the detection range of CPT-11 of one of the metabolites is 40-12000ng/mL, and the lower limit mass number of the quantification is 40 ng/mL; one of the metabolites of SN38 was detected in the range of 0.1-30. mu.g/mL, with a lower mass limit of quantitation of 0.1. mu.g/mL.
The invention has the beneficial effects that:
the invention establishes an analysis method which has good selectivity and high separation degree and can simultaneously and quantitatively determine n-valent PEG irinotecan prodrug and metabolites with different forms in vivo in a biological sample
The invention is based on the biological mass spectrum full spectrum scanning (MS)ALL) Technique, polydisperse PEG- (CPT-11)n(wherein n is 2-12) and PEG- (CPT-11)n(m)(m-1, 2 … 11) is broken into specific fragment ions with lower molecular weight and stable signal intensity, and the fragment ion pair PEG- (CPT-11) is characterized by m/z 569.2751 for the first timenAnd PEG- (CPT-11)n(m)Go on to decideAnd (4) analyzing the quantity. The PEG- (CPT-11) which can simultaneously separate and quantify the n-valent PEG irinotecan prodrug is established for the first timenAnd analysis method of in vivo different form metabolite. Effectively breaks through PEG- (CPT-11)nA technical bottleneck of polymorphic ingredient in vivo fate research.
The invention realizes PEG- (CPT-11) for the first time by groping the liquid phase conditionnWith PEG- (CPT-11)n(m)The chromatography of (3) is effective.
The invention is described in detail below with reference to the figures and examples.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 is PEG-PEGylated irinotecan prodrug PEG- (CPT-11)3(A) CPT-11(B), SN38 (C);
FIG. 2 is PEG-PEGylated irinotecan prodrug PEG- (CPT-11)3Fragmentation at impact energy CE-50;
FIG. 3 is PEG- (CPT-11)3、PEG-(CPT-11)3(2)、PEG-(CPT-11)3(1)(A) Typical chromatograms of PEG non-drug moiety (B), CPT-11(C) and SN38 (D);
FIG. 4 shows example 1 PEG- (CPT-11) PEG prodrug of PEGylated irinotecan in rat tail vein3The plasma drug concentration-time curve of each component to be tested;
FIG. 5 is the PEG-prodrug of irinotecan of example 2 injected into the tail vein of rats (CPT-11)3The cumulative excretion rate of each test component in urine versus time curve.
Detailed Description
Example 1
This example specifically measures trivalent pegylated irinotecan prodrug and its metabolites of different morphologies in vivo in rat plasma.
PEG- (CPT-11) is weighed precisely3About 500mg of standard substance, after the purity is converted, the standard substance is dissolved by sodium chloride solution for injection and diluted to 50mg/mL, the rat is administrated with 120mg/kg in a single tail vein, and the blood sampling time points are as follows: 5min, 10min, 30min, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h and 48 h. MeasuringDetermination of PEG- (CPT-11) in plasma after tail vein administration in rats3And the content of metabolites with different forms, the plasma drug concentration time curve is shown in FIG. 4.
Determination of PEG- (CPT-11)3The method mainly comprises the following steps:
a1, pretreatment of a plasma sample:
1) add 50. mu.L of biological sample (anticoagulant when plasma samples are collected) to the polyethylene tube,
2) adding 50 μ L of internal standard, mixing by vortex,
3) protein precipitation: adding 250 mu L of acetonitrile, mixing evenly by vortex,
4) is centrifuged at 13000rpm for 10 minutes,
5) transferring 50 mu L of centrifuged supernatant into a 2mL EP tube containing 950 mu L of purified water, and mixing uniformly by vortex;
b1, preparing a standard curve:
1) PEG- (CPT-11) was extracted from blank rat plasma3The stock solutions are respectively diluted to 3.0, 6.0, 10.0, 30.0, 100.0, 300.0 and 500.0 mu g/mL to be used as a standard curve I;
2) preparing an internal standard working solution: the internal standard diazepam was diluted to 2.0 μ g/mL internal standard working solution using methanol-acetonitrile-water (1/1/2, v/v).
3) Taking 20 μ l for liquid chromatography-tandem mass spectrometry, recording chromatogram, and using PEG- (CPT-11)3The concentration is the abscissa, the peak area ratio of the analyte to the internal standard is the ordinate, and the weight W is 1/x2And performing regression operation by a least square method to obtain a linear regression equation. Determination of PEG- (CPT-11) in rat plasma3The linear range of the working curve of the concentration is 3.0-500 mug/mL, and the LLOQ is 3.0 mug/mL.
C1, preparing a quality control sample:
preparation of PEG- (CPT-11) according to the protocol of "preparation of Standard Curve3The concentrations of the low, medium and high samples are 5.0, 50.0 and 400.0 mu g/mL, the concentration of each sample is obtained by controlling the quality of the sample, and the concentration of each sample is obtained according to a standard curve.
Determination of PEG- (CPT-11)3(2)、PEG-(CPT-11)3(1)PEG non-drug moieties, irinotecan and SNThe main steps of 38 are as follows:
a2, pretreatment of a plasma sample:
1) 30 mul of the biological sample was added to a polyethylene tube,
2) adding 30 μ L of internal standard, mixing by vortex,
3) protein precipitation: adding 150 mu L of acetonitrile, mixing evenly by vortex,
4) centrifugation is carried out at 13000rpm for 10 minutes,
5) transferring 100 mu L of centrifuged supernatant into a 2mL EP tube containing 100 mu L of aqueous solution, and mixing uniformly by vortex;
b2, preparing a standard curve:
1) PEG- (CPT-11)3(2)、PEG-(CPT-11)3(1)The stock solutions of PEG20K, CPT-11 and SN38 are prepared into a mixed stock solution according to a certain proportion. Gradually diluting the mixed stock solution with blank rat plasma, and gradually diluting with PEG- (CPT-11)3(2)Respectively diluting the stock solution to 5.0, 15.0, 25.0, 50.0, 75.0, 150.0 and 250.0 mu g/mL; PEG- (CPT-11)3(1)Respectively diluting the stock solution to 1.0, 3.0, 5.0, 10.0, 15.0, 30.0 and 50.0 mu g/mL; the concentration of PEG20K is 1.0, 3.0, 5.0, 10.0, 15.0, 30.0 and 50.0 mu g/mL in sequence; the concentration of irinotecan is 20.0, 60.0, 100.0, 200.0, 300.0, 600.0 and 1000.0ng/mL in sequence; the SN38 concentrations are 10.0, 30.0, 50.0, 100.0, 150.0, 300.0 and 500.0ng/mL in sequence, and are taken as a standard curve II;
2) preparing an internal standard working solution: the internal standard diazepam was diluted to 200.0ng/mL of internal standard working solution using methanol-acetonitrile-water (1/1/2, v/v).
3) Taking 20 μ l of the extract, performing liquid chromatography-tandem mass spectrometry, recording chromatogram, and analyzing with PEG- (CPT-11)3(2)、PEG-(CPT-11)3(1)PEG20K, irinotecan and SN38 were used as abscissa, peak area ratio of corresponding analyte and internal standard was used as ordinate, and weight W was 1/x2And performing regression operation by a least square method to obtain a linear regression equation. Determination of PEG- (CPT-11) in rat plasma3(2)、PEG-(CPT-11)3(1)The linear ranges of the working curves of the PEG20K, the irinotecan and the SN38 are respectively 5.0-250 mu g/mL, 1.0-50 mu g/mL, the concentration is 1.0-50.0 mu g/mL, 20.0-1000.0ng/mL, 10.0-500.0ng/mL, and LLOQ of 5.0. mu.g/mL, 1.0. mu.g/mL, 20.0ng/mL, and 10.0ng/mL, respectively.
C2, preparing a quality control sample:
preparation of PEG- (CPT-11) according to the protocol of "preparation of Standard Curve3(2)、PEG-(CPT-11)3(1)Low, medium and high concentrations of PEG20K, irinotecan and SN38 (12.5, 40, 200 μ g/mL, 2.5, 8.0, 37.5 μ g/mL, 50.0, 160.0, 750.0ng/mL, 25.0, 80.0, 375.0ng/mL) quality control samples, at least three samples per concentration, giving the concentrations according to the standard curve.
The conditions measured by the quantitative method in the above steps are as follows:
the chromatographic conditions are as follows: a chromatographic column: agilent 5TC-C18(2) column, 250mm × 4.6mm, 5 μm particle size; mobile phase: water phase: aqueous, organic phase containing 0.1% formic acid: 50% acetonitrile + 50% methanol (containing 0.1% formic acid); gradient elution; the column temperature is 50 ℃; the flow rate is 1.0 mL/min; the sample size was 20. mu.L, and the total run time was 20 min.
The gradient elution in the chromatographic conditions described, the procedure is shown in Table 1,
TABLE 1 gradient elution procedure
Figure BDA0003603146340000081
The mass spectrum conditions are as follows: triple TOFTM6600+A tandem mass spectrometer of the type equipped with an ESI ionization source; detecting in a positive ion mode; ion spray voltage 5500V; the temperature is 550 ℃; source gas 1: nitrogen pressure 50 psi; gas 2: nitrogen pressure 50 psi; gas curtain gas: nitrogen pressure 40 psi; cluster-splitting voltage: 50V; MS (Mass Spectrometry)ALLThe scanning range of the scanning mode is 50-2500; the scanning range of the Product Ion scanning mode is 200-400; PEG- (CPT-11)3、PEG-(CPT-11)3(2)、PEG-(CPT-11)3(1)The collision energy of the channel was 50eV, the collision energy of the PEG non-drug moiety and the irinotecan channel was 30eV, and the collision energy of the SN38 channel was 40 eV.
In view of PEG- (CPT-11)3And its in vivo different form metabolic product typicalLinearity of the Standard Curve and the drug concentration-time curves of the various morphological constituents measured in plasma (FIG. 4), the simultaneous analysis of PEG- (CPT-11) in plasma according to the invention3And the method for separating metabolites with different forms in vivo has good chromatographic separation effect, the biomass spectrum quantitative method has good linear relation, the accuracy, the precision and the like completely accord with the relevant guiding principles of FDA and CFDA biological sample analysis methods, and can be used for PEG- (CPT-11) in plasma3And synchronous quantitative analysis of metabolites with different forms in vivo.
Example 2
This example specifically measures trivalent pegylated irinotecan prodrug and its metabolites of different morphologies in vivo in rat urine.
The rat is subjected to 120mg/kg single tail vein administration, and the urine of the rat is collected at 4h, 12h, 24h, 36h, 48h, 60h, 72h, 96h, 120h, 144h, 168h, 192h, 216h, 240h, 264h, 288h, 312h and 336 h. Determination of PEG- (CPT-11) in rat urine3And the content of metabolites with different forms in the body, and calculating the cumulative excretion rate of each form component in the urine of the rat. The cumulative rate of excretion of urine versus time curve for the rats is shown in FIG. 5.
The method mainly comprises the following steps:
A. pretreating a urine sample:
1) 50 μ L of the biological sample was added to a polyethylene tube,
2) adding 50 μ L of internal standard, mixing by vortex,
3) precipitating protein: adding 250 mu L of acetonitrile, mixing evenly by vortex,
4) centrifugation is carried out at 13000rpm for 10 minutes,
5) transferring 150 μ L of centrifuged supernatant into a 2mL EP tube containing 150 μ L of purified water (1/1, v/v), and mixing by vortexing;
B. preparation of a standard curve:
1) PEG- (CPT-11) was extracted from white rat urine3Stock solutions were diluted to 1.0, 30.0, 50.0, 100.0, 300.0, 500.0, 2000.0 μ g/mL, respectively, as standard curve I; mixing PEG- (CPT-11)3(2)、PEG-(CPT-11)3(1)The stock solutions of PEG20K, irinotecan and SN38 are mixed according to a certain proportionAnd (4) stock solution. Diluting the mixed stock solution with blank rat urine step by step, PEG- (CPT-11)3(2)The concentration is 3, 9, 15, 30, 90, 300 and 900 mu g/mL in sequence; PEG- (CPT-11)3(1)The concentration is 1.0, 3.0, 5.0, 10.0, 30.0, 100.0 and 300.0 mu g/mL in sequence; the concentration of PEG20K is 1.0, 3.0, 5.0, 10.0, 30.0, 100.0 and 300.0 mu g/mL in sequence; the irinotecan concentration is 40.0, 120.0, 200.0, 400.0, 1200.0, 4000.0 and 12000.0ng/mL in sequence; the SN38 concentrations are 0.1, 0.3, 0.5, 1.0, 3.0, 10.0 and 30.0 mu g/mL in sequence, and are taken as a standard curve II;
2) preparing an internal standard working solution: the internal standard diazepam was diluted to 500ng/mL internal standard working solution using methanol-acetonitrile-water (1/1/2, v/v).
3) Taking 10 mul to perform liquid chromatography-tandem mass spectrometry, recording chromatogram, taking the concentration of each shape to-be-detected substance as an abscissa, taking the peak area ratio of the to-be-detected substance and the internal standard as an ordinate, and using the weighted W as 1/x2And performing regression operation by a least square method to obtain a linear regression equation. Determination of PEG- (CPT-11) in rat urine3The linear range of the working curve of (1.0-2000.0) mu g/mL, PEG- (CPT-11)3(2)The linear range of the working curve is 3.0-900.0 mu g/mL, PEG- (CPT-11)3(1)And the linear range of the working curve of the PEG20K is 1.0-300.0 mu g/mL; the linear range of the working curve of the irinotecan is 40.0-12000.0 ng/mL; the linear range of the working curve of SN38 is 0.1-30.0. mu.g/mL.
C. Preparation of quality control samples:
operating under the "preparation of Standard Curve", PEG- (CPT-11)3The concentrations of the low, the middle and the high are 2.5, 25 and 1600 mu g/mL respectively, PEG- (CPT-11)3(2)、PEG-(CPT-11)3(1)The low, medium and high concentrations of PEG20K, irinotecan and SN38 (7.5, 24, 720 μ g/mL, 2.5, 8.0, 240 μ g/mL, 2.5, 8, 240.0 μ g/mL, 100.0, 320.0, 9600.0ng/mL, 0.25, 0.8, 24.0 μ g/mL) quality control samples were at least three samples per concentration and the concentrations were determined from the standard curve.
The conditions measured by the quantitative method in the above steps are as follows:
the chromatographic conditions are as follows: a chromatographic column: agilent 5TC-C18(2) column, 250mm multiplied by 4.6mm, 5 μm particle size; mobile phase: water phase: aqueous, organic phase containing 0.1% formic acid: 50% acetonitrile + 50% methanol (containing 0.1% formic acid); gradient elution; the column temperature is 50 ℃; the flow rate is 1.0 mL/min; the gradient elution procedure with a sample size of 10. mu.L and a total run time of 20min is shown in Table 1.
The mass spectrum conditions are as follows: triple TOFTM6600+A tandem mass spectrometer of the type equipped with an ESI ionization source; detecting in a positive ion mode; ion spray voltage 5500V; the temperature is 550 ℃; source gas 1: nitrogen pressure 50 psi; gas 2: nitrogen pressure 50 psi; gas curtain gas: nitrogen pressure 40 psi; cluster-splitting voltage: 50V; MS (Mass Spectrometry)ALLThe scanning range of the scanning mode is 50-2500; the scanning range of the Product Ion scanning mode is 200-400; PEG- (CPT-11)n3、PEG-(CPT-11)n3(m2)、PEG-(CPT-11)n3(m1)The collision energy of the channel was 50eV, the collision energy of the PEG non-drug moiety and the irinotecan channel was 30eV, and the collision energy of the SN38 channel was 40 eV.
In view of PEG- (CPT-11)3The linearity of typical standard curves of metabolites with different forms in vivo and the drug concentration of each form component in the urine are measured, the invention synchronously analyzes PEG- (CPT-11) in the urine3And the method for separating metabolites with different forms in the body has good chromatographic separation effect, the biomass spectrum quantitative method has good linear relation, the accuracy, the precision and the like completely accord with the relevant guiding principles of FDA and CFDA biological sample analysis methods, and the method can be used for PEG- (CPT-11) in urine3And synchronous quantitative analysis of metabolites with different forms in vivo.
The above-mentioned embodiments are only part of the present invention, and do not cover the whole of the present invention, and on the basis of the above-mentioned embodiments and the attached drawings, those skilled in the art can obtain more embodiments without creative efforts, so that the embodiments obtained without creative efforts are all included in the protection scope of the present invention.

Claims (7)

1. A quantitative analysis method for multivalent PEGylated irinotecan prodrug and metabolites thereof in biological samples is characterized by comprising the following steps of: measuring by high performance liquid chromatography-time of flight mass spectrometry by combining a full spectrum scanning mode of biological mass spectrometry with a scanning mode of daughter ions; grouping and separating the n-valent PEGylated irinotecan prodrug and the mixture of metabolites with different forms in vivo in a biological sample by high performance liquid chromatography; selecting common characteristic fragments through mass spectrometry, and quantifying each component;
the n-valent PEGylated irinotecan prodrug is expressed as PEG- (CPT-11)n(ii) a Wherein n represents a valence state, and means a PEG- (CPT-11)nThe maximum value of the parent drug is loaded on the prodrug molecule, and n is 2-12;
the mixture of metabolites of different morphologies in vivo of n-valent pegylated irinotecan prodrug comprises: PEGylated irinotecan prodrug metabolite PEG- (CPT-11) of different drug loading statesn(m)Each component, dissociated irinotecan CPT-11, irinotecan active metabolite SN38 and PEG non-drug part;
wherein, PEG- (CPT-11)n(m)In the method, m is the number of the residual CPT-11 residues on PEG, and m is 1-11;
wherein, the time-of-flight mass spectrum selects PEG- (CPT-11)nAnd PEG- (CPT-11)n(m)Consensus characteristic fragment m/z 569.27, characteristic fragment m/z 587.28 of CPT-11, characteristic cleavage reaction fragment m/z 393.15 → 349.14 of SN38, characteristic fragment m/z 177.11 of a PEG non-drug moiety grouping.
2. The method for quantitative analysis of multivalent pegylated irinotecan prodrug and its metabolites in biological samples according to claim 1, characterized in that: the specific mass spectrometry flow is as follows: 1) MS (Mass Spectrometry)ALLIn the scanning mode, all the objects to be detected pass through the Q1, the objects to be detected can be smashed through collision in the Q2 process, and the TOF mass analyzer scans all passing ions to obtain resolution information of all fragments; under the passage of CE50, PEG- (CPT-11) was extracted from the obtained fragmentsnWith PEG- (CPT-11)n(m)Common characteristic fragment 569.27 + -0.02 Da, characteristic fragment 177.11 + -0.02 Da of PEG non-drug part extracted under CE30 channel, and characteristic fragment 587.28 + -0.02 Da of irinotecan extracted; 2) in Product Ion scan mode, CE was set to 40, SN38 quantified Ion pairs as m/z 393.15→349.14。
3. The method of claim 1, wherein the method comprises the steps of: liquid chromatography conditions: using a reverse phase chromatography column, aqueous phase: water containing 0.1% formic acid; organic phase: 50% acetonitrile + 50% methanol, containing 0.1% formic acid; the gradient elution started with a 10% organic phase proportion and subsequently transitioned to 70% with a maximum organic phase proportion of 95%.
4. The method of claim 1, wherein the method comprises the steps of: mass spectrum conditions: a series quadrupole time-of-flight mass spectrum, an ESI ionization source; the acquisition mode adopts MSALLCombining a Product Ion scanning mode; detecting positive ions; ion spray voltage 5000-; the temperature is 450 ℃ and 550 ℃; source gas 1: nitrogen pressure 40 + -10 psi; gas 2: nitrogen pressure 40 + -10 psi; gas curtain gas: nitrogen pressure 40-55 psi; cluster-splitting voltage: 50V; MS (Mass Spectrometry)ALLThe scanning range of the scanning mode is 50-2500 Da; the scanning range of the Product Ion scanning mode is 200-400 Da; the collision energy is 10-50 eV; wherein, PEG irinotecan prodrug PEG- (CPT-11)nAnd PEG-modified irinotecan prodrug metabolite PEG- (CPT-11) in different drug loading statesn(m)The PEG non-drug part and the CPT-11 adopt MSALLA scanning mode; SN38 uses Product Ion scanning mode.
5. The method of claim 1, wherein the method comprises the steps of: the biological sample is plasma, urine, bile or feces.
6. The method of claim 1, wherein the method comprises the steps of: before the determination, the biological sample is subjected to pretreatment and a preparation step of a standard curve; determining the supernatant subjected to pretreatment by using high performance liquid chromatography-time-of-flight mass spectrometry, performing full-spectrum scanning and conjugant ion scanning analysis, and recording a chromatogram; substituting peak areas of each component of the n-valent PEGylated irinotecan prodrug and metabolites with different forms in vivo into a standard curve to obtain respective corresponding concentrations.
7. The method of claim 1, wherein the method comprises the steps of: the pretreatment operations of different samples are as follows: adding anticoagulant and precipitated protein into the plasma sample, centrifuging, and taking supernatant; precipitating protein in urine and bile samples, centrifuging, and taking supernate; freeze drying fecal sample, crushing, re-dissolving, vortex centrifuging, taking supernatant, precipitating protein, centrifuging and diluting supernatant.
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