CN115078621A - Method for determining concentration of irinotecan derivative Dxd in blood plasma - Google Patents
Method for determining concentration of irinotecan derivative Dxd in blood plasma Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 40
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- 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 determining the concentration of an irinotecan derivative Dxd in blood plasma, which is used for detecting the concentration of a blood plasma sample by high performance liquid chromatography-tandem mass spectrometry after pretreatment, and comprises the following steps: s1, pretreatment of a plasma sample: taking a plasma sample in a 96-deep-hole plate, adding acetonitrile, mixing in a vortex manner, centrifuging, taking supernatant liquid until complex solution H is added 2 O/1M CH 3 COONH 4 Vortex mixing in a 96-deep well plate of (1), H 2 O and 1M CH 3 COONH 4 Is 100/0.1, as a test sample to be detected; s2, sample measurement: the test sample was injected into a high performance liquid chromatography-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 determining the blood concentration of Dxd.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a method for determining a medicine, and particularly relates to a method for determining the concentration of an irinotecan derivative Dxd in blood plasma.
Background
Irinotecan derivative (Dxd) is a potent DNA topoisomerase I inhibitor and is useful as a payload for antibody-conjugated drug ADC (DS-8201a) 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-1H, 12H-benzo (de) pyrano (3',4',6,7) indolizino (1,2-b) quinolin-1-yl) -2-hydroxyacetamide, formula: c 26 H 24 FN 3 O 6 Molecular weight: 493.48, having the chemical formula:
dxd As a small molecule poison for coupling ADC drugs, only the first three-common drug in the market abroad and Enhertu developed by Aslicon are currently applied to China in 2022 and are included in the priority review stage. The breast cancer is used as the malignant tumor with the highest incidence rate of Chinese females, T-DXd fills the unmet treatment requirement of HER2 positive late stage, and an innovative treatment situation is developed for Chinese HER2 positive breast cancer patients. The first one is that the drug-loaded target is precisely transferred to cancer cell tissues through a unique linker technology by means of the originally developed Dxd technical advantages, so that a more stable, efficient and safe treatment means is brought to patients. Several pharmaceutical companies developed several pharmaceutical products for conjugation Dxd to act on HER 2.
Dxd has strong toxicity, and has strong killing effect on normal cells of human body during anti-tumor therapy, and since ADC drug has strong technical limitation in the research and development process, it has very important effect on free Dxd drug detection in both new drug research and development stage and drug clinical monitoring stage after drug is on the market.
Due to the targeting property of the ADC drug, the ADC drug enters cells through endocytosis, the free concentration is extremely low, the requirement on a method for detecting the free Dxd is high, meanwhile, the Dxd special chemical structure has not very strong hydrophilic/hydrophobic effect, the chromatographic retention has a challenge, in addition, a lactone ring exists in the compound, the stability has a great problem, and the stability in the processes of sample collection, transportation, storage and detection is ensured through method development. Therefore, it is necessary to establish a method which is fast, accurate and reliable, and 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 irinotecan derivative Dxd in plasma that solves one or more of the above-mentioned problems of the prior art.
The invention provides a method for determining concentration of an irinotecan derivative Dxd in blood plasma, which comprises the following steps of:
s1, pretreatment of a plasma sample: taking a plasma sample in a 96-deep-hole plate, adding acetonitrile, mixing in a vortex manner, centrifuging, taking supernatant liquid until complex solution H is added 2 O/1M CH 3 COONH 4 Vortex mixing in a 96-deep well plate of (1), H 2 O and 1M CH 3 COONH 4 Is 100/0.1, and is used as a test sample to be detected;
s2, sample measurement: the test sample was injected into a high performance liquid chromatography-tandem mass spectrometer, the chromatographic peak of Dxd in the sample was detected, and Dxd concentration in the plasma sample was calculated therefrom.
In certain embodiments, the plasma sample in step S1 further comprises a pH adjustment prior to placing in the 96-deep well plate, and the pH is adjusted to 6.0.
In certain embodiments, the plasma sample in step S1 is expressed in K 2 EDTA is anticoagulant.
In certain embodiments, the plasma sample is added to acetonitrile in step S1, vortexed for 2min, and centrifuged at 2600g for 10min at 4 ℃; adding a complex solution H 2 O/1M CH 3 COONH 4 Vortex mixing for 2min, H 2 O and 1M CH 3 COONH 4 The volume ratio of (A) to (B) is 100/0.1.
In certain embodiments, the liquid chromatography assay conditions in step S2 are:
a chromatographic column: GL Sciences, InertSustain, C18 HP, 3 μm, 2.1X 50mm (UP);
temperature of the chromatographic column: 40 ℃;
mobile phase A: h with a 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 O solution;
washing liquid: ACN/H with volume ratio of 85/15/0.2 2 O/FA solution;
the temperature of the autosampler is 4 ℃;
gradient elution, flow rate of 0.5mL/min, sample size of 10. mu.L, analysis time of 3.205 min.
In certain embodiments, the mass spectrometry conditions in step S2 are:
the ion source adopts an electrospray ion source, the spraying voltage is 5500V, the atomizing temperature is 550 ℃, the spraying air pressure is 60Psi, the auxiliary heating air pressure is 60Psi, the air curtain air pressure is 25Psi, the collision air pressure is 8Psi, and the declustering voltage is 60 eV;
the collision cell inlet voltage is 10 eV;
the collision voltage is 40 eV;
the outlet voltage of the collision chamber is 10 eV;
detecting in a positive ion mode;
the scanning mode is multiple reaction detection;
dxd the ion pairs used for quantitative analysis were: m/z494.3 → m/z 419.1; the ion pairs for qualitative analysis were: m/z494.3 → m/z 375.3.
In certain embodiments, the procedure for gradient elution 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 by substituting the peak area of Dxd into a standard curve equation.
In certain embodiments, the establishing of the standard curve equation comprises the steps of:
taking 8 parts of 190 mu L blank plasma (pH is 6.0), placing the blank plasma in a 1.5mL centrifuge tube, adding Dxd solutions with the concentrations of 400pg/mL, 800pg/mL, 2000pg/mL, 8000pg/mL, 32000pg/mL, 80000pg/mL, 320000pg/mL and 4000000pg/mL in the form of stock solution to a standard sample 1, a standard sample 2, a standard sample 3, a standard sample 4, a standard sample 5, a standard sample 6, a standard sample 7 and a standard sample 8, respectively taking the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6, the standard sample 7, the standard sample 8 and a zero-concentration sample 50 mu L in a 96 deep-well plate, adding 200 mu L acetonitrile, vortex mixing for 2min, centrifuging for 10min at 4 ℃ by 2600g, and taking the upper layer100 mu L of clear liquid to 100 mu L of compound solution H 2 O/1M CH 3 COONH 4 ，H 2 O and 1M CH 3 COONH 4 Mixing in a 96 deep-hole plate with the volume ratio of 100/0.1 for 2min in a vortex mode to serve as a test sample to be detected;
each 10 μ L of the test sample was injected into a high performance liquid chromatography-tandem mass spectrometer, and a chromatographic peak of Dxd in the sample was detected, from which a standard curve was obtained for calculating the concentration of Dxd in the plasma (pH 6.0).
Has the advantages that: the method for determining the concentration of the irinotecan derivative Dxd in the plasma has the following advantages:
(1) the pretreatment method is simple and convenient, one-step organic solvent protein precipitation is adopted, and the method is suitable for conventional determination;
(2) the specificity is strong: under the chromatographic conditions adopted in the experiment, the Dxd retention time is about 1.140min, and the peak pattern of Dxd is good;
(3) the sensitivity is high: the minimum limit of quantitation of the plasma is 20.0pg/mL, the concentration of Dxd in the plasma can be accurately determined, the sensitivity is high, and the specificity is strong;
(4) the method 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 linear range of the plasma standard curve of the method is 20.0-20000pg/mL, and the precision RSD in and among batches is less than +/-15%.
Drawings
FIG. 1 is a standard curve of Dxd in human plasma measured by HPLC-MS/MS method according to the present invention;
FIG. 2 is a graph of HPLC-MS/MS of the present inventors' blank plasma addition Dxd;
FIG. 3 is a HPLC-MS/MS chart of the blank plasma of the present invention.
Detailed Description
The present invention will be described in further detail below with reference to embodiments.
EXAMPLE 1 Experimental materials and analytical Equipment
Dxd (analyte): GLPBIO or same, higher grade standard
The reagents used are shown in table 1 below:
TABLE 1 details of reagents
Note: the same or higher level of reagents may also be used
The analytical equipment used is shown in table 2 below:
TABLE 2 details of the devices used
| Assembly | Species of | Manufacturer(s) |
| Binary pump (Binary pump) | AD Pump | SHIMADZU |
| Degasser (deaerator) | Degasser | SHIMADZU |
| Column oven (constant temperature Column box) | AD Column oven | SHIMADZU |
| Autosampler (automatic sampler) | AC Autosampler | SHIMADZU |
| Sample rack | Rack Changer | SHIMADZU |
| Mass spectrometer | TRIPLE QUAD 6500+ | SCIEX |
| Data processor | Analyst(software) | SCIEX |
The same LC-MS/MS system may also be used.
Example 2 liquid phase conditions
1. Conditions of liquid chromatography
A chromatographic column: GL Sciences, InertSustain, C18 HP, 3 μm, 2.1X 50mm (UP); temperature of the chromatographic column: 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 is 85/15/0.2; the temperature of the autosampler is 4 ℃; gradient elution with flow rate of 0.5mL/min, sample size of 10 μ L, and analysis time of 3.205 min;
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. Conditions of Mass Spectrometry
The ion source adopts an electrospray ion source, the spraying voltage is 5500V, the atomizing temperature is 550 ℃, the spraying air pressure is 60Psi, the auxiliary heating air pressure is 60Psi, the air curtain air pressure is 25Psi, the collision air pressure is 8Psi, and the declustering voltage is 60 eV; the collision cell inlet voltage is 10 eV; the collision voltage is 40 eV; the outlet voltage of the collision chamber is 10 eV; detecting in a positive ion mode; the scanning mode is multiple reaction detection;
dxd the ion pairs used for quantitative analysis were: m/z494.3 → m/z 419.1; and (3) qualitative analysis of ion pairs: m/z494.3 → m/z 375.3.
EXAMPLE 3 Linear test
1. Dxd preparation of Standard solution
Dxd preparation of standard solution: dxd (analyte) 100 mu g is precisely weighed, 50% acetonitrile is dissolved to 100 mu g/mL, and 50% acetonitrile water solution is sequentially diluted to prepare Dxd standard solution, wherein the specific dilution concentration is shown in the following Table 4:
TABLE 4 Dxd Standard solution preparation concentrations
| Source solution (pg/mL) | Volume of source solution (μ L) | Volume of solvent (μ L) | 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 the standard solution was stored in brown glass containers and refrigerators (-20 ℃) when not in use, with the volume being scaled up or down as required.
3. Linear test
Unfreezing the blank plasma in a water bath at room temperature, and adjusting the pH value to 6.0 by using citric acid; 10 portions of 190 μ L of blank plasma were transferred to a 96-well plate (each standard curve sample, blank sample-00 and zero concentration sample-0), and according to the following table 5, 10 μ L of Dxd standard solution or diluted solution with different concentrations was precisely added to prepare each sample and mixed to prepare drug-containing plasma with different concentrations, and the operation was performed according to "pretreatment of plasma sample". The peak area value Y was used for the regression calculation of the blood concentration X, and the results are shown in FIG. 1 and Table 6. The blood concentration X is subjected to regression calculation by Y to obtain a regression equation Y which is 691000X-770, the correlation coefficient (R) is 0.9996, and the fitting degree (R) is 2 ) Is 0.99920016. Weight coefficient W is 1/X 2 The lowest quantitative limit of Dxd plasma concentration measured by this method is: 20.0pg/mL, quantitative lower line STD1 representative profile is shown in FIG. 2.
TABLE 5 Dxd Standard Curve formulation concentrations
a: diluted solution of analyte: 50% aqueous acetonitrile solution
TABLE 6 Standard Curve for HPLC-MS/MS Dxd in human plasma (n-5)
Example 4 accuracy and precision
Unfreezing the blank plasma in a water bath at room temperature, and adjusting the pH value to 6.0 by using citric acid; transfer appropriate volume of blank plasma to appropriate container and add Dxd standard solution to prepare 5 kinds of drug-containing plasma quality control samples (LLOQ QC, LQC, GMQC, MQC, HQC) with different concentrations and a following standard curve, operate according to "plasma sample pretreatment", quality control sample preparation is as shown in Table 7 below. Making one batch and one following standard curve every day, continuously making 5 batches for 5 days, respectively making 6 samples of each concentration of the first batch, the second batch, the third batch, the fourth batch and the fifth batch, calculating Dxd peak area, substituting into the standard curve of the day to obtain the actually measured concentration, calculating the precision between batches according to the actually measured concentration, wherein the ratio of the actually measured concentration to the theoretical concentration is the accuracy, and the result is shown in Table 8. Dxd the plasma sample precision and accuracy between batches and within batches are within plus or minus 15 percent.
TABLE 7 quality control sample preparation concentration
Sufficient volume was dispensed into the labeled sample vial and stored at the theoretical temperature-20 ℃ as required for each assay batch. The volume may be scaled up or down as desired.
TABLE 8 HPLC-MS/MS method for determining Dxd accuracy and precision within and between batches of plasma (pH 6.0)
Example 5 interference
The interference of different blank plasma with Dxd analytes was evaluated by using 6 blank plasma samples from different sources, 3 hyperlipidemia plasma from different sources and 3 hemolytic plasma from different sources for the same assay batch prepared and analyzed according to the sample preparation procedure.
After the 12 blank plasma samples from different sources are prepared and analyzed, the interference peak responses at the retention time meeting Dxd are all lower than 20.0% of the Dxd response of the quantitative lower limit sample in the standard curve of the analysis batch, the results are shown in Table 9, and the interference representative pattern of the blank plasma on the analyte is shown in FIG. 3. The results show that this analysis method is specific to the analysis at Dxd.
TABLE 912 interference data of blank plasma from different sources for Dxd analytes
As can be seen from table 9, blank plasma from different human bodies did not interfere with the detection result of Dxd. Thus, the method can be used to detect Dxd concentrations in different human plasmas.
Example 6 recovery
Analyte recovery will be calculated using LQC, MQC and HQC formulated with pooled plasma (PH 6.0). Extraction was performed for 6 parallel samples and 18 DBs each of LQC, MQC and HQC. The analytes were added after DB extraction, so that their concentration in the DB extract added after extraction was the same as the 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 area between ± 15.0% at each concentration level of the analyte, and a recovery of 103.4%, indicating a high extraction recovery using this pretreatment method Dxd.
TABLE 10 extraction recovery of 10 Dxd analytes
Example 7 matrix Effect
Blank matrix samples from at least 6 different sources were processed. The analytes were added after extraction of DB to ensure that their concentration in the processed DB (1 sample per source per concentration) was the same as the processed LQC, MQC and HQC samples; solutions containing the analytes were prepared to final concentrations identical to the treated LQC, MQC and HQC samples, 6 replicates per concentration, with results in table 11, analyte peak area ratios% CVs all between ± 15%, indicating the absence of matrix effect problems.
TABLE 11 matrix Effect of 11 Dxd analytes
In summary, the following steps: the invention provides a method for measuring Dxd concentration in plasma (PH is 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 free from the interference of miscellaneous peaks, and the base line is stable; the method has high specificity, can accurately determine the concentration of Dxd in plasma (PH is 6.0), has high sensitivity, and has the minimum limit of plasma quantification of 20.0 pg/mL; meanwhile, 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. The linear range of the plasma standard curve of the method is 20.0-20000pg/mL, the precision RSD in batches and among batches is +/-15%, the extraction recovery rate of analytes is high, and the matrix effect does not exist.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these should be considered as within the scope of the present invention.
Claims (9)
1. A method for determining concentration of an irinotecan derivative Dxd in plasma is characterized in that the concentration of the plasma sample is detected by high performance liquid chromatography-tandem mass spectrometry after the plasma sample is pretreated, and the specific method comprises the following steps:
s1, pretreatment of plasma samples: taking a plasma sample in a 96-deep-hole plate, adding acetonitrile, mixing in a vortex manner, centrifuging, taking supernatant liquid until complex solution H is added 2 O/1M CH 3 COONH 4 Vortex mixing in a 96-deep well plate of (1), H 2 O and 1M CH 3 COONH 4 Is 100/0.1, and is used as a test sample to be detected;
s2, sample measurement: the test sample was injected into a high performance liquid chromatography-tandem mass spectrometer, the chromatographic peak of Dxd in the sample was detected, and the Dxd concentration in the plasma sample was calculated therefrom.
2. The method of claim 1, wherein the step of adjusting the pH of the plasma sample in step S1 before being placed in a 96-well plate is further performed by adjusting the pH to 6.0.
3. A method of determining the concentration of irinotecan derivative Dxd in plasma according to claim 2Wherein the plasma sample is expressed as K in step S1 2 EDTA is anticoagulant.
4. The method for determining the concentration of Dxd irinotecan derivative in blood plasma as claimed in claim 2, wherein the plasma sample is vortexed with acetonitrile at 4 ℃ for 2min and centrifuged at 2600g for 10min at S1; adding a complex solution H 2 O/1M CH 3 COONH 4 Vortex mixing for 2min, H 2 O and 1M CH 3 COONH 4 The volume ratio of (A) to (B) is 100/0.1.
5. The method for determining the concentration of an irinotecan derivative Dxd in blood plasma according to claim 2, characterized in that the conditions of the liquid chromatography determination in step S2 are as follows:
and (3) chromatographic column: GL Sciences, InertSustain, C18 HP, 3 μm, 2.1X 50mm (UP);
temperature of the chromatographic column: 40 ℃;
mobile phase A: h with a volume ratio of 100/0.2 2 O/1M CH 3 COONH 4 A solution;
and (3) mobile phase B: ACN/H with volume ratio of 95/5 2 O solution;
washing liquid: ACN/H with volume ratio of 85/15/0.2 2 An O/FA solution;
the temperature of the automatic sample injector is 4 ℃;
gradient elution, flow rate of 0.5mL/min, sample size of 10. mu.L, analysis time of 3.205 min.
6. The method for determining the concentration of an irinotecan derivative Dxd in blood plasma according to claim 2, wherein the mass spectrometry conditions in step S2 are as follows:
the ion source adopts an electrospray ion source, the spraying voltage is 5500V, the atomizing temperature is 550 ℃, the spraying air pressure is 60Psi, the auxiliary heating air pressure is 60Psi, the air curtain air pressure is 25Psi, the collision air pressure is 8Psi, and the declustering voltage is 60 eV;
the inlet voltage of the collision chamber is 10 eV;
the collision voltage is 40 eV;
the outlet voltage of the collision chamber is 10 eV;
detecting in a positive ion mode;
the scanning mode is multiple reaction detection;
dxd the ion pairs used for quantitative analysis were: m/z494.3 → m/z 419.1; the ion pairs for qualitative analysis were: m/z494.3 → m/z 375.3.
7. The method of claim 5, wherein the gradient elution is performed by a procedure comprising:
8. The method of claim 2, wherein the concentration of irinotecan derivative Dxd in the plasma is calculated by substituting the peak area of Dxd into a standard curve equation in step S2 to calculate the concentration of Dxd in the plasma sample.
9. The method of claim 8, wherein the standard curve equation is established by the steps of:
putting 8 parts of 190 mu L blank plasma (pH 6.0) into a 1.5mL centrifuge tube, adding Dxd solutions with the concentrations of 400pg/mL, 800pg/mL, 2000pg/mL, 8000pg/mL, 32000pg/mL, 80000pg/mL, 320000pg/mL and 4000000pg/mL respectively into a standard sample 1, a standard sample 2, a standard sample 3, a standard sample 4, a standard sample 5, a standard sample 6, a standard sample 7 and a standard sample 8 in the form of stock solution, respectively taking the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6, the standard sample 7, the standard sample 8 and a zero-concentration sample 50 mu L into a 96 deep-well plate, adding 200 mu L acetonitrile, mixing for 2min in a vortex manner, centrifuging for 10min at 4 ℃ by 2600g, taking 100 mu L of supernatant to 100 mu L of complex solution H added with the H100 mu L complex solution 2 O/1M CH 3 COONH 4 ，H 2 O and 1M CH 3 COONH 4 Mixing in a 96 deep-hole plate with the volume ratio of 100/0.1 for 2min in a vortex mode to serve as a test sample to be detected;
10 μ L of each test sample was injected into a hplc-tandem mass spectrometer, and a chromatographic peak of Dxd in the sample was detected, from which a standard curve was obtained for calculating the concentration of Dxd in the plasma (pH 6.0).
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