CN115078621B - Method for measuring concentration of irinotecan derivative Dxd in blood plasma - Google Patents
Method for measuring concentration of irinotecan derivative Dxd in blood plasma Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 34
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical class C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 title claims abstract description 13
- 210000002381 plasma Anatomy 0.000 title abstract description 50
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000012360 testing method Methods 0.000 claims abstract description 12
- 238000002156 mixing Methods 0.000 claims abstract description 11
- 238000005259 measurement Methods 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 239000006228 supernatant Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 19
- 238000001514 detection method Methods 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 239000007921 spray Substances 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 5
- 238000000889 atomisation Methods 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000004811 liquid chromatography Methods 0.000 claims description 3
- 238000004949 mass spectrometry Methods 0.000 claims description 3
- 238000004451 qualitative analysis Methods 0.000 claims description 3
- 238000004445 quantitative analysis Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 2
- 239000003146 anticoagulant agent Substances 0.000 claims description 2
- 229940127219 anticoagulant drug Drugs 0.000 claims description 2
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims description 2
- 239000011550 stock solution Substances 0.000 claims 2
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- 229940049595 antibody-drug conjugate Drugs 0.000 description 5
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
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Abstract
The invention discloses a method for measuring the concentration of an irinotecan derivative Dxd in blood plasma, which comprises the following steps of: s1, pretreatment of plasma samples: taking plasma sample in 96 deep hole plate, adding acetonitrile, vortex mixing, centrifuging, collecting supernatant, adding complex solution H 2 O/1M CH 3 COONH 4 Vortex mixing in 96 deep well plate, H 2 O and 1M CH 3 COONH 4 The volume ratio of (2) is 100/0.1, and the sample is used as a test sample to be detected; s2, sample measurement: the test sample was injected into a high performance liquid chromatograph-tandem mass spectrometer, the chromatographic peak of Dxd in the sample was detected, and the Dxd concentration in the plasma sample was calculated therefrom. The method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of Dxd.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a method for measuring the concentration of an irinotecan derivative Dxd in blood plasma.
Background
Irinotecan derivative (Dxd) is a potent DNA topoisomerase I inhibitor and can be used as a payload for antibody-conjugated drug ADC (DS-8201 a) targeting HER 2. Its chemical name N- ((1S, 9S)9-ethyl-5-fluoro-2,3,9,10,13,15-hexahydro-9-hydroxy-4-methyl-10, 13-dioxo-1 h,12 h-benzo (de) pyrano (3 ',4',6, 7) indolizino (1, 2-b) quinolin-1-yl) -2-hydroxyacetamide of formula: c (C) 26 H 24 FN 3 O 6 Molecular weight: 493.48, the chemical structural formula is:
dxd is used as a coupled small molecule poison of ADC drugs, only the first three commonly marketed at home and recently developed Enhertu by Ashikan, and was applied home in 2022 and incorporated into the priority review stage. The breast cancer is taken as the malignant tumor with highest incidence rate of Chinese females, T-DXd fills the treatment requirement which is not met in the advanced stage of HER2 positive, and the novel treatment situation is developed for Chinese HER2 positive breast cancer patients. The first three uses the original Dxd technical advantages to accurately transfer the drug-carrying targeting to the cancer cell tissues through the unique linker technology, thereby bringing more stable, efficient and safe treatment means for patients. There are also several research and development pharmaceutical enterprises in China that develop ADC drugs that act on HER2 conjugated Dxd.
Dxd has strong toxicity, has strong killing effect on normal cells of human body during anti-tumor treatment, has extremely important effect on free Dxd drug detection, whether in a new drug research and development stage or after drug marketing, because ADC drugs have strong technical limitations in the research and development process.
Due to the targeting of ADC drugs, the free concentration is extremely low by endocytosis into cells, the requirements for methods for detecting free Dxd are high, the specific chemical structure of Dxd is not very strong, the hydrophilic/hydrophobic effects of the compounds are not very strong, chromatographic retention is challenging, in addition, a lactone ring exists in the compounds, the stability is very problematic, and the stability during sample collection, transportation, storage and detection is ensured by the development of the methods. It is therefore desirable to establish a method that is fast, accurate, reliable, and at the same time has high sensitivity and high selectivity.
Disclosure of Invention
It is an object of the present invention to provide a method for determining the concentration of an irinotecan derivative Dxd in plasma which solves one or more of the above mentioned problems of the prior art.
The invention provides a method for measuring the concentration of an irinotecan derivative Dxd in blood plasma, which comprises the following steps of:
s1, pretreatment of plasma samples: taking plasma sample in 96 deep hole plate, adding acetonitrile, vortex mixing, centrifuging, collecting supernatant, adding complex solution H 2 O/1M CH 3 COONH 4 Vortex mixing in 96 deep well plate, H 2 O and 1M CH 3 COONH 4 The volume ratio of (2) is 100/0.1, and the sample is used as a test sample to be detected;
s2, sample measurement: the test sample was injected into a high performance liquid chromatograph-tandem mass spectrometer, the chromatographic peak of Dxd in the sample was detected, and the Dxd concentration in the plasma sample was calculated therefrom.
In certain embodiments, the plasma sample of step S1 further comprises a pH adjustment prior to placement in the 96-well plate, and the pH is adjusted to 6.0.
In certain embodiments, the plasma sample in step S1 is in K 2 EDTA is an anticoagulant.
In certain embodiments, the plasma sample in step S1 is added to acetonitrile for a vortex mixing time of 2min and centrifuged at 2600g for 10min at 4 ℃; adding the complex solution H 2 O/1M CH 3 COONH 4 Vortex mixing 2min, H 2 O and 1M CH 3 COONH 4 The volume ratio of (2) is 100/0.1.
In certain embodiments, the liquid chromatography assay conditions in step S2 are:
chromatographic column: GL Sciences, inertSustain, C18 HP,3 μm, 2.1X150 mm (UP);
chromatographic column temperature: 40 ℃;
mobile phase a: h with volume ratio of 100/0.2 2 O/1M CH 3 COONH 4 A solution;
mobile phase B: ACN/H with volume ratio of 95/5 2 An O solution;
washing liquid: ACN/H with volume ratio of 85/15/0.2 2 An O/FA solution;
the temperature of the automatic sampler is 4 ℃;
gradient elution was carried out at a flow rate of 0.5mL/min, a sample injection amount of 10. Mu.L, and an analysis time of 3.205min.
In certain embodiments, the mass spectrometry conditions in step S2 are:
the ion source adopts an electrospray ion source, the spray voltage is 5500V, the atomization temperature is 550 ℃, the spray gas pressure is 60Psi, the auxiliary heating gas pressure is 60Psi, the gas curtain gas pressure is 25Psi, the collision gas pressure is 8Psi, and the cluster removal voltage is 60eV;
the collision cell inlet voltage is 10eV;
the collision voltage is 40eV;
the collision cell exit voltage is 10eV;
detecting a positive ion mode;
the scanning mode is multiple reaction detection;
dxd ion pairs for quantitative analysis are: m/z494.3 →m/z419.1; the ion pairs for qualitative analysis were: m/z494.3 →m/z375.3.
In certain embodiments, the gradient elution procedure is:
| total time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0.10 | 70 | 30 |
| 1.40 | 5 | 95 |
| 2.00 | 5 | 95 |
| 2.02 | 70 | 30 |
| 3.20 | 70 | 30 |
In certain embodiments, the concentration of Dxd in the plasma sample is calculated in step S2 with the peak area of Dxd being taken into the standard curve equation.
In certain embodiments, the establishment of the standard curve equation comprises the steps of:
8 parts of 190 mu L blank plasma (pH=6.0) are placed in a 1.5mL centrifuge tube, 10 mu L acetonitrile solution with concentration of 400pg/mL, 800pg/mL, 2000pg/mL, 8000pg/mL, 32000pg/mL, 80000pg/mL, 32000pg/mL and 4000000pg/mL is added to a sample 1, a sample 2, a sample 3, a sample 4, a sample 5, a sample 6, a sample 7 and a sample 8 respectively, a sample 1, a sample 2, a sample 3, a sample 4, a sample 5, a sample 6, a sample 7, a sample 8 and a sample 50 mu L with zero concentration are respectively added to a 96 deep hole plate, 200 mu L acetonitrile is added to be vortex mixed for 2min, centrifugation is carried out at 2600g for 10min at 4 ℃, and the upper layer of 100 mu L is added to 100 mu L complex solution H is obtained 2 O/1M CH 3 COONH 4 ，H 2 O and 1M CH 3 COONH 4 Vortex mixing for 2min in a 96 deep hole plate with the volume ratio of 100/0.1 to be used as a test sample to be detected;
10 μl of each test sample was injected into a high performance liquid chromatograph-tandem mass spectrometer, the chromatographic peak of Dxd in the sample was detected, and a standard curve was obtained therefrom for calculating the concentration of Dxd in the plasma (ph=6.0).
The beneficial effects are that: the method for measuring the concentration of the irinotecan derivative Dxd in the blood plasma has the following advantages:
(1) The pretreatment method is simple and convenient, and the one-step organic solvent protein precipitation is suitable for routine determination;
(2) The specificity is strong: under the chromatographic conditions adopted in the experiment, the retention time of Dxd is about 1.140min, and the peak type of Dxd is good;
(3) The sensitivity is high: the minimum quantitative limit of the plasma is 20.0pg/mL, the concentration of Dxd in the plasma can be accurately measured, the sensitivity is high, and the specificity is strong;
(4) The method provided by the invention is rapid, accurate, high in precision and sensitivity, and simple and convenient to operate, and provides a basis for measuring the blood concentration of Dxd. The plasma standard curve of the method has a linear range of 20.0-20000pg/mL, and the precision RSD in and between batches is less than + -15%.
Drawings
FIG. 1 shows a standard curve of Dxd in human plasma measured by HPLC-MS/MS method of the present invention;
FIG. 2 is a HPLC-MS/MS diagram of a blank plasma addition Dxd of the present invention;
FIG. 3 is an HPLC-MS/MS diagram of the blank plasma of the present inventors.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments.
Example 1 Experimental materials and analytical apparatus
Dxd (analyte): GLPBIO or the same, higher grade standard
The reagents used are shown in Table 1 below:
TABLE 1 details of reagents
Note that: the same or higher grade reagents may also be used
The analytical equipment was used as shown in table 2 below:
table 2 usage device details
| Assembly | Species of type | Manufacturer (S) |
| Binary pump (Binary pump) | AD Pump | SHIMADZU |
| Degasser (Degasser) | Degasser | SHIMADZU |
| Column cover (constant temperature Column box) | AD Column oven | SHIMADZU |
| Autosampler (Autosampler) | AC Autosampler | SHIMADZU |
| Sample rack (Sample rack) | Rack Changer | SHIMADZU |
| Mass spectrometer (Mass spectrometer) | TRIPLE QUAD 6500+ | SCIEX |
| Data processor (Data processor) | Analyst(software) | SCIEX |
The same LC-MS/MS system may also be used.
EXAMPLE 2 liquid phase Condition
1. Conditions of liquid chromatography
Chromatographic column: GL Sciences, inertSustain, C18 HP,3 μm, 2.1X150 mm (UP); chromatographic column temperature: 40 ℃; mobile phase a: h 2 O/1M CH 3 COONH 4 =100/0.2; mobile phase B: ACN/H 2 O=95/5; washing liquid: ACN/H 2 O/fa=85/15/0.2; the temperature of the automatic sampler is 4 ℃; gradient elution, the flow rate is 0.5mL/min, the sample injection amount is 10 mu L, and the analysis time is 3.205min;
TABLE 3 gradient elution procedure
| Total time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0.10 | 70 | 30 |
| 1.40 | 5 | 95 |
| 2.00 | 5 | 95 |
| 2.02 | 70 | 30 |
| 3.20 | 70 | 30 |
2. Mass spectrometry conditions
The ion source adopts an electrospray ion source, the spray voltage is 5500V, the atomization temperature is 550 ℃, the spray gas pressure is 60Psi, the auxiliary heating gas pressure is 60Psi, the gas curtain gas pressure is 25Psi, the collision gas pressure is 8Psi, and the cluster removal voltage is 60eV; the collision cell inlet voltage is 10eV; the collision voltage is 40eV; the collision cell exit voltage is 10eV; detecting a positive ion mode; the scanning mode is multiple reaction detection;
dxd ion pairs for quantitative analysis are: m/z494.3 →m/z419.1; qualitative analysis of ion pairs: m/z494.3 →m/z375.3.
Example 3 Linear test
1. Preparation of Dxd Standard solution
Preparing Dxd standard solution: dxd (analyte) 100 μg was precisely weighed, 50% acetonitrile water was dissolved to 100 μg/mL,50% acetonitrile water was dissolved and diluted in sequence to prepare Dxd standard solution, and specific diluted concentrations are shown in table 4 below:
TABLE 4 Dxd standard solution formulation concentration
| Source solution (pg/mL) | Source solution volume (μL) | Solvent volume (μL) | Final concentration (pg/mL) |
| 100000000 | 20 | 1980 | 1000000 |
| 1000000 | 800 | 1200 | 400000 |
| 1000000 | 640 | 1360 | 320000 |
| 1000000 | 160 | 1840 | 80000 |
| 320000 | 200 | 1800 | 32000 |
| 320000 | 50 | 1950 | 8000 |
| 32000 | 125 | 1875 | 2000 |
| 32000 | 50 | 1950 | 800 |
| 32000 | 25 | 1975 | 400 |
Dxd standard solution is stored in brown glass container and refrigerator (-20 deg.C) when not in use, and the volume can be increased or decreased proportionally as required.
3. Linearity test
Thawing blank plasma in water bath at room temperature, and regulating pH to 6.0 with citric acid; 10 parts of 190 mu L of blank plasma are transferred to a 96-deep well plate (each standard curve sample, blank sample-00 and zero concentration sample-0), 10 mu L of Dxd standard solution or diluted solution with different concentrations are respectively and precisely added to prepare each sample according to the following table 5, and the samples are uniformly mixed to prepare medicine-containing plasma with different concentrations, and the operation of plasma sample pretreatment is performed. Regression calculation was performed on the blood concentration X with the peak area value Y, and the results are shown in FIG. 1 and Table 6. Regression calculation is performed on the blood concentration X by Y to obtain a regression equation Y= 691000X-770, the correlation coefficient (R) is 0.9996, and the fitting degree (R 2 ) 0.99920016. Weight coefficient w=1/X 2 The lowest quantitative limit of the blood concentration of Dxd measured by this method is: a representative map of quantitative off-line STD1 at 20.0pg/mL is shown in FIG. 2.
TABLE 5 Dxd standard curve formulation concentration
a: diluted solution of analyte: 50% acetonitrile aqueous solution
Table 6 standard curve of Dxd in human plasma (n=5) by HPLC-MS/MS method
Example 4 accuracy and precision
Thawing blank plasma in water bath at room temperature, and regulating pH to 6.0 with citric acid; transfer appropriate volumes of blank plasma to appropriate containers and add Dxd standard solution to prepare 5 different concentrations of drug-containing plasma quality control samples (LLOQ QC, LQC, GMQC, MQC, HQC) and a follow-up standard curve, prepared as shown in table 7 below, following a "plasma sample pretreatment" procedure. And (3) carrying out one batch and one follow-up standard curve every day, continuously carrying out 5 days, carrying out 5 batches in total, respectively carrying out 6 samples on each concentration of the first batch, the second batch, the third batch, the fourth batch and the fifth batch, calculating Dxd peak area, substituting the peak area into the standard curve of the current day to obtain actual measurement concentration, calculating the precision between batches in the batch according to the actual measurement concentration, wherein the ratio of the actual measurement concentration to the theoretical concentration is the accuracy, and the result is shown in Table 8.Dxd plasma sample batch-to-batch, intra-batch precision and accuracy are within + -15% and meet the requirements.
TABLE 7 quality control sample formulation concentrations
A sufficient volume was dispensed into labeled sample vials and stored at-20 ℃ as required for each assay batch. The volume may be scaled up or down as desired.
Table 8 HPLC-MS/MS method to determine in-batch, inter-batch and accuracy of Dxd in plasma (ph=6.0)
EXAMPLE 5 interference
The interference of different blank plasma on Dxd analytes was evaluated by using 6 blank plasma samples of different sources, 3 high fat plasma of different sources and 3 hemolyzed plasma of different sources in the same assay batch prepared and analyzed according to the sample preparation procedure.
After preparation of 12 blank plasma samples from different sources, the response of the interference peak at the retention time corresponding to Dxd was lower than 20.0% of the Dxd response of the lower limit sample in the standard curve of the assay batch, the results are shown in table 9, and a representative profile of interference of blank plasma on analyte is shown in fig. 3. The results indicate that the analysis method is specific to the analysis of Dxd.
Table 9 comparison of interference data from 12 different sources of blank plasma against Dxd analyte
As can be seen from table 9, the blank plasma from different human bodies did not interfere with the Dxd detection results. Thus, the method can be used to detect Dxd concentrations in different human plasma.
EXAMPLE 6 recovery
The recovery of analyte will be calculated using LQC, MQC and HQC formulated with pooled plasma (ph=6.0). For each of 6 parallel samples of LQC, MQC and HQC and 18 DB were extracted. Analytes were added after DB extraction, so that their concentration in DB extract added after extraction was the same as that of extracted LQC, MQC and HQC samples. The extraction recovery was calculated by comparing the peak areas of the analyte and internal standard from QC with the average peak areas of the analyte and internal standard added after DB extraction, and the results are shown in table 10, with% CV and overall% CV of the analyte peak areas between ± 15.0% for each concentration level of analyte, and a recovery of 103.4%, indicating high extraction recovery using this pretreatment method Dxd.
TABLE 10 extraction recovery of Dxd analytes
EXAMPLE 7 matrix Effect
Blank matrix samples from at least 6 different sources were treated. Analytes were added after extraction of DB, ensuring their concentration in post-treatment DB (1 sample per source per concentration) was the same as the post-treatment LQC, MQC and HQC sample concentrations; solutions containing analytes were prepared to have the same final concentration as the concentrations of the treated LQC, MQC and HQC samples, 6 in parallel, and the results are shown in table 11, with the analyte peak area ratio%cv between ± 15%, indicating no matrix effect problem.
TABLE 11 matrix Effect of Dxd analytes
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To sum up: the invention provides a method for measuring Dxd concentration in plasma (PH=6.0) with simple pretreatment method, which adopts a one-step organic solvent precipitation method and is suitable for conventional measurement; meanwhile, under the chromatographic condition adopted in the experiment, the retention time of Dxd is about 1.140min, the peak shape is good, the measurement is not interfered by the impurity peak, and the baseline is stable; the method has higher specificity, can accurately measure the concentration of Dxd in the blood plasma (PH=6.0), has higher sensitivity, and the lowest quantitative limit of the blood plasma is 20.0pg/mL; meanwhile, the method is quick, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of Dxd. The plasma standard curve linear range of the method is 20.0-20000pg/mL, the precision RSD in the batch and between batches is within +/-15%, the analyte extraction recovery rate is high and no matrix effect exists.
The above description is only of a preferred form of the invention, it being noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the inventive concept, which shall also be regarded as being within the scope of the invention.
Claims (4)
1. A method for measuring the concentration of an irinotecan derivative Dxd in plasma, which is characterized in that the concentration of a plasma sample is detected by high performance liquid chromatography-tandem mass spectrometry after pretreatment, and the specific method comprises the following steps: s1, pretreatment of plasma samples: taking plasma sample in 96 deep hole plate, adding acetonitrile, vortex mixing, centrifuging, collecting supernatant, adding complex solution H 2 O/1M CH 3 COONH 4 Vortex mixing in 96 deep well plate, H 2 O and 1M CH 3 COONH 4 The volume ratio of (2) is 100/0.1, and the sample is used as a test sample to be detected; s2, sample measurement: injecting a test sample into a high performance liquid chromatography-tandem mass spectrometer, detecting a chromatographic peak of Dxd in the sample, and calculating Dxd concentration in the plasma sample therefrom;
the plasma sample in the step S1 is placed in front of the 96 deep-hole plate and further comprises the adjustment of the pH value, and the pH value is adjusted to 6.0;
the liquid chromatography measurement conditions in step S2 are: chromatographic column: GL Sciences, inertSustain, C18 HP,3 μm, 2.1X150 mm; chromatographic column temperature: 40 ℃; mobile phase a: h with volume ratio of 100/0.2 2 O/1M CH 3 COONH 4 A solution; mobile phase B: ACN/H with volume ratio of 95/5 2 An O solution; washing liquid: ACN/H with volume ratio of 85/15/0.2 2 An O/FA solution; the temperature of the automatic sampler is 4 ℃; gradient elution, the flow rate is 0.5mL/min, the sample injection amount is 10 mu L, and the analysis time is 3.205min;
the gradient elution procedure was:
；
the mass spectrometry conditions in step S2 are: the ion source adopts an electrospray ion source, the spray voltage is 5500V, the atomization temperature is 550 ℃, the spray gas pressure is 60Psi, the auxiliary heating gas pressure is 60Psi, the gas curtain gas pressure is 25Psi, the collision gas pressure is 8Psi, and the cluster removal voltage is 60eV; the collision cell inlet voltage is 10eV; the collision voltage is 40eV; the collision cell exit voltage is 10eV; detecting a positive ion mode; the scanning mode is multiple reaction detection; dxd ion pairs for quantitative analysis are: m/z494.3 →m/z419.1; the ion pairs for qualitative analysis were: m/z494.3 →m/z375.3.
2. The method for determining the concentration of irinotecan derivative Dxd in plasma according to claim 1, characterized in that the plasma sample in step S1 is in K 2 EDTA is an anticoagulant.
3. A method of determining the concentration of an irinotecan derivative Dxd in plasma according to claim 1, characterized in that the concentration of Dxd in the plasma sample is calculated in step S2 with Dxd peak area carry-over into standard curve equation.
4. A method for determining the concentration of an irinotecan derivative Dxd in plasma according to claim 3, characterized in that the establishment of the standard curve equation comprises the steps of: taking 8 parts of 190 mu L blank plasma, placing into a 1.5mL centrifuge tube, adding 10 mu L of Dxd solution with concentration of 400pg/mL, 800pg/mL, 2000pg/mL, 8000pg/mL, 32000pg/mL, 80000pg/mL, 32000pg/mL and 4000000pg/mL into a 1.5mL centrifuge tube, 2.2, 3.4.5.6.7.8 stock solution, respectively taking 1.2.3.4.5.6.7.8 stock solution, 50 mu L zero concentration sample into a 96 deep well plate, vortex mixing for 2min, adding 200 mu L acetonitrile into a 10min at 4 ℃, centrifuging at 2600g, and taking 100 mu L upper layer clear solution into 100 mu L complex solution H 2 O/1M CH 3 COONH 4 ，H 2 O and 1M CH 3 COONH 4 Vortex mixing in 96 deep hole plate with volume ratio of 100/0.1 for 2minAs a test sample to be detected; 10. Mu.L of each test sample was injected into a high performance liquid chromatograph-tandem mass spectrometer, the chromatographic peak of Dxd in the sample was detected, and a standard curve was obtained therefrom for calculating the concentration of Dxd in the plasma.
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