CN112684057A - Kit for detecting concentration of 11 anti-tumor drugs in serum and application thereof - Google Patents
Kit for detecting concentration of 11 anti-tumor drugs in serum and application thereof Download PDFInfo
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Abstract
The invention discloses a kit for detecting the concentration of 11 anti-tumor drugs in serum and application thereof, wherein the kit comprises eluent, calibrator solution, internal standard solution, protein precipitator and quality control product, and belongs to the technical field of blood detection. By adopting the kit, the concentration of the 11 anti-tumor drugs in the serum of a human body can be simultaneously determined by an ultra-high performance liquid chromatography-tandem mass spectrometry (ID-HPLC-MS/MS) technology, the serum dosage is small (only 50 muL) and the pretreatment is simple, and the analysis of multiple substances by one needle only needs 6.5 minutes, so that the kit is simple and rapid, the accuracy and the precision basically meet the requirements, and the kit can be used for the quantitative analysis of the concentrations of the 11 anti-tumor drugs in the serum clinically and provides a simple and rapid detection method for the monitoring of the concentrations of the 11 anti-tumor drugs clinically.
Description
Technical Field
The invention belongs to the technical field of blood detection, and particularly relates to a kit for detecting the concentration of 11 anti-tumor drugs in serum and application thereof.
Background
At present, malignant tumor is one of the major diseases threatening human life and health, and the disease has the characteristics of high morbidity, high lethality rate and the like. The cancer morbidity and mortality of China in the last 70 th century are higher, namely the gastric cancer, the esophageal cancer, the liver cancer, the cervical cancer and the lung cancer, and the liver cancer and the lung cancer rise to the first few in the 90 th century. In recent years, according to statistics of 7 large areas of north China, northeast China, east China, south China, southwest China and northwest China, 10 people in China have lung cancer, stomach cancer, liver cancer, esophageal cancer and the like of men and breast cancer, lung cancer, colorectal cancer, gastric cancer and the like of women before the incidence rate of tumors. Moreover, with the aging of population, the incidence of cancer in China is gradually increased, and cancer is the first cause of death of residents in China at present, thus not only forming a great threat to health, but also causing serious burden to the development of the economic society. Due to the high incidence of cancer, the combination of drugs is increasing, and therefore, the monitoring of the blood concentration of cancer drugs is of great significance.
Capecitabine is suitable for late stage primary or metastatic breast cancer, is rapidly absorbed by intestinal mucosa after being taken orally, is converted into inactive intermediate 5 '-deoxy-5' fluorocytidine by carboxyl esterase in liver, is converted into 5 '-deoxy-5' fluorouridine by the action of cytidine deaminase of liver and tumor tissues, and is catalyzed into fluorouracil (5-FU) by thymidine phosphorylase to act in tumor tissues. Afatinib is a new generation of oral small molecule Tyrosine Kinase Inhibitors (TKIs), is the first irreversible ErbB family blocker, can act on the whole ErbB family including EGFR, and is different from the first generation of reversible EGFR TKIs, afatinib can be irreversibly combined with EGFR, so that the purposes of closing cancer cell signaling pathways and inhibiting tumor growth are achieved. Tamoxifen is a synthetic antiestrogen. The structure of the estrogen-like compound is similar to that of estrogen, and the estrogen-like compound can compete with estradiol for estrogen receptors, form stable complexes with the estrogen receptors, and transport the stable complexes into the nucleus to prevent chromosome genes from opening, so that the growth and development of cancer cells are inhibited. Gefitinib is an oral epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitor (a small molecule compound), and the inhibition of EGFR-TK can block the growth, metastasis and angiogenesis of tumors and increase the apoptosis of tumor cells. Imatinib, a tyrosine kinase inhibitor, is a small molecule protein kinase inhibitor, has the function of blocking one or more protein kinases, and is clinically used for treating chronic myelogenous leukemia and malignant gastrointestinal stromal tumors. Sunitinib is a novel multi-targeted oral drug for treating tumors, gastrointestinal stromal tumors and metastatic renal cell carcinoma that are not responsive or tolerated by standard therapies. Erlotinib is a targeted therapeutic drug, can specifically act on tumor cells, inhibits the formation and growth of tumors, and inhibits the growth of tumors by inhibiting the activity of tyrosine kinase, which is one of important components in EGFR cells, and is used for three-line treatment of locally advanced or metastatic non-small cell lung cancer with failure of two or more chemotherapy schemes. The hydrochloric acid Icotinib is the first small molecule targeted anticancer drug in China, is a high-efficiency and specific epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), and is suitable for second-line treatment of advanced non-small cell lung cancer. The apatinib is suitable for treating advanced gastric adenocarcinoma or adenocarcinoma of the gastroesophageal junction by three or more lines. Crizotinib is a tyrosine kinase receptor inhibitor, comprising ALK, hepatocyte growth factor receptor (HGFR, c-Met), ROS1(c-cos) and RON, translocation can cause the ALK gene to cause the expression of oncogenic fusion protein, and the formation of the ALK fusion protein can cause the activation and the imbalance of gene expression and signals, thereby causing the proliferation and the survival of tumor cells expressing the proteins.
At present, the method for detecting the concentration of the antitumor drug mainly adopts a high performance liquid chromatography-tandem mass spectrometry method, for example, patent CN108828077A discloses a kit for simultaneously detecting capecitabine and its metabolites in plasma, and a detection method and an application thereof, and the method has certain defects: firstly, only one drug and its metabolite are detected, and the clinical significance is limited because most patients are combined with other drugs; secondly, the quantitative off-line of capecitabine in the method is high, and the method has certain limitation on medication guidance. For another example, An article entitled "An LC-MS/MS method for rapid and positive high-throughput simultaneous determination of human plasma kinase inhibitors in human plasma" detects 5 protein kinase inhibitor drugs based on LC-MS/MS, the sample dosage of the method is 300 μ L, and the pretreatment adopts more complicated liquid-liquid extraction method, even if so, the lower limit of the quantitation of the afatinib is 5ng/mL, and the trough concentration after clinical afatinib administration is lower than 5ng/mL, so the application in clinic is very limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the kit for detecting the concentrations of the 11 anti-tumor drugs in the serum, and the kit can be used for simply, efficiently and reliably detecting the concentrations of the 11 anti-tumor drugs in the serum at the same time, and can further meet the requirements of clinical application.
In order to achieve the purpose, the invention adopts the following technical scheme:
a kit for detecting the concentration of 11 antitumor drugs in serum comprises: eluent, calibrator solution, internal standard solution, protein precipitator and quality control product;
the 11 anti-tumor drugs are: capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib;
the eluent comprises an eluent A and an eluent B, wherein the eluent A is a mixed solution of 0.01-0.2% formic acid and 1 mM-3 mM ammonium acetate water solution, and the eluent B is acetonitrile;
the calibrator solution comprises a mixed solution of a series of known concentrations of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib prepared from a blank serum matrix;
the internal standard solution is a methanol water solution containing Afatinib-d 6, apatinib-d 8, gefitinib-d 3, imatinib-d 8, sunitinib-d 4, mu-de-ethylsunitinib-d 5, tamoxifen-d 5, capecitabine-d 11 and crizotinib-d 5;
the protein precipitator is a mixed solution of methanol and acetonitrile;
the quality control product is a mixed solution of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-dethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib which are prepared from a blank serum matrix and have known concentration.
By adopting the kit, the concentration of the 11 anti-tumor drugs in the human serum can be simultaneously determined by an ultra-high performance liquid chromatography-tandem mass spectrometry (ID-HPLC-MS/MS) technology, the serum dosage is less (only 50 mu L), the pretreatment is simple, and the analysis of various substances by one needle only needs 6.5 minutes, so that the kit is simple and rapid.
Has the advantages that: when the kit provided by the invention is used for detecting the concentration of the 11 anti-tumor drugs in the serum, the sensitivity is high, the specificity is strong, the accuracy is high, the pretreatment process is simple, the separation and the detection of the 11 anti-tumor drugs in the serum are completed within 6.5 minutes, the accuracy and the precision basically meet the requirements, the kit can be used for the quantitative analysis of the 11 anti-tumor drugs in the serum clinically, and a simple and rapid detection method is provided for the concentration monitoring of the 11 anti-tumor drugs clinically.
Drawings
FIG. 1 is an ion flowgram of 11 antitumor drug standards;
FIG. 2 is an ion flow chart of 11 kinds of antitumor drugs extracted from serum.
Detailed Description
The invention provides a kit for detecting the concentration of 11 anti-tumor drugs in serum, which can realize the simultaneous detection of the concentration of the 11 anti-tumor drugs in the serum by an ultra-high performance liquid chromatography tandem mass spectrometry technology.
The 11 anti-tumor drugs are: capecitabine (CPT), Afatinib (AFT), Tamoxifen (TMX), Gefitinib (GFT), Imatinib (IMT), Sunitinib (SNT), Erlotinib (ELT), N-desethylsunitinib (NSNT), erlotinib hydrochloride (ICT), Apatinib (APT), and Crizotinib (CZP);
the internal standard substances corresponding to the 11 antitumor drugs are as follows: capecitabine-d 11(CPT-d11), Afatinib-d 6(AFT-d6), tamoxifen-d 5(TMX-d5), gefitinib-d 3(GFT-d3), imatinib-d 8(IMT-d8), sunitinib-d 4(SNT-d4), N-desethylsunitinib-d 5(NSNT-d5), apatinib-d 8(APT-d8), and crizotinib-d 5(CZP-d 5);
the kit comprises the following reagents:
(1) eluent:
eluent A: 0.01 to 0.2 percent of formic acid and 1 to 3mM of ammonium acetate aqueous solution; eluent B: acetonitrile;
(2) calibration solution:
standard stock solutions containing Afatinib 4 μ g/mL, Apatinib 8 μ g/mL, gefitinib 40 μ g/mL, erlotinib hydrochloride 20 μ g/mL, crizotinib 10 μ g/mL, imatinib 40 μ g/mL, sunitinib 4 μ g/mL, N-desethylsunitinib 4 μ g/mL, erlotinib 80 μ g/mL, tamoxifen 40 μ g/mL, and capecitabine 20 μ g/mL were formulated in a blank serum matrix as seven calibrator solutions at different concentration points:
the seven concentration points of afatinib, sunitinib and N-dethylsunitinib are as follows in sequence: 0.4ng/mL, 1ng/mL, 2ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 200 ng/mL;
the seven concentration points of apatinib are in sequence: 0.8ng/mL, 2ng/mL, 4ng/mL, 20ng/mL, 40ng/mL, 200ng/mL, 400 ng/mL;
seven concentration points of crizotinib are in order: 1ng/mL, 2.5ng/mL, 5ng/mL, 25ng/mL, 50ng/mL, 250ng/mL, 500 ng/mL;
the seven concentration points of the icotinib hydrochloride and capecitabine are as follows: 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000 ng/mL;
seven concentration points of gefitinib, imatinib and tamoxifen are as follows: 4ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000 ng/mL;
seven concentration points of erlotinib are in order: 8ng/mL, 20ng/mL, 40ng/mL, 200ng/mL, 400ng/mL, 2000ng/mL, 4000 ng/mL;
(3) internal standard solution:
an aqueous methanol solution comprising Afatinib-d 65 μ g/mL, Apatinib-d 82 μ g/mL, gefitinib-d 32 μ g/mL, imatinib-d 85 μ g/mL, sunitinib-d 40.4 μ g/mL, μm-desethylsunitinib-d 50.4 μ g/mL, tamoxifen-d 50.5 μ g/mL, capecitabine-d 111 μ g/mL, and crizotinib-d 52 μ g/mL;
(4) protein precipitant:
a mixed solution of methanol and acetonitrile;
(5) quality control product:
blank serum matrix containing 11 kinds of antitumor drugs with low, medium and high concentrations of QC (L), QC (M) and QC (H), wherein,
qc (l) was a 5000-fold dilution of the above standard stock solution in blank serum matrix.
Qc (m) is a 500-fold dilution of the above standard stock solution in blank serum matrix.
Qc (h) was diluted 50-fold with blank serum matrix for the standard stock solution described above.
In a preferred embodiment, the eluent A is a mixed solution of 0.01% -0.2% formic acid and 1 mM-3 mM ammonium acetate solution, preferably a mixed solution of 0.1% formic acid and 2mM ammonium acetate solution.
The methanol water is 40-60% methanol water solution; preferably, the aqueous methanol is 50% aqueous methanol.
In one scheme, the volume ratio of methanol to acetonitrile in the protein precipitator is 1: 1-3; preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 2.
In one embodiment, the serum blank matrix is serum blank without the drug of interest.
The standard stock solutions mentioned in the present invention were prepared as follows:
the above standards were formulated at the following concentrations: afatinib 0.1mg/mL, Apatinib 0.1mg/mL, gefitinib 5mg/mL, hydrochloric acid of Icotinib 2mg/mL, crizotinib 0.1mg/mL, imatinib 2mg/mL, sunitinib 0.1mg/mL, N-ethyl sunitinib 0.1mg/mL, erlotinib 2mg/mL and tamoxifen 2mg/mL, capecitabine 2 mg/mL;
transferring a standard product mother solution: afatinib 20 μ L, apatinib 40 μ L, gefitinib 4 μ L, erlotinib hydrochloride 5 μ L, crizotinib 50 μ L, imatinib 10 μ L, sunitinib 20 μ L, N-desethylsunitinib 20 μ L, erlotinib 20 μ L, tamoxifen 10 μ L, and capecitabine 5 μ L; then added to 296. mu.L of 50% methanol water to give 500. mu.L of a standard stock solution.
The mixed internal standard solution mentioned in the invention is prepared according to the following method:
the following internal standard mother liquor was prepared: afatinib-d 61 mg/mL, apatinib-d 81 mg/mL, gefitinib-d 31mg/mL, imatinib-d 81 mg/mL, sunitinib-d 40.1mg/mL, N-desethylsunitinib-d 50.1mg/mL, tamoxifen-d 50.1mg/mL, capecitabine-d 110.1mg/mL and crizotinib-d 51 mg/mL;
transferring internal standard mother liquor: afatinib-d 62.5 μ L, apatinib-d 81 μ L, gefitinib-d 31 μ L, imatinib-d 82.5 μ L, sunitinib-d 42 μ L, N-desethylsunitinib-d 52 μ L, tamoxifen-d 52.5 μ L, capecitabine-d 115 μ L, and crizotinib-d 51 μ L; then adding into 480.5 μ L50% methanol water to obtain 500 μ L internal standard solution; wherein Afatinib-d 65000ng/mL, Apatinib-d 82000 ng/mL, gefitinib-d 32000 ng/mL, imatinib-d 85000 ng/mL, sunitinib-d 4400 ng/mL, N-desethylsunitinib-d 5400 ng/mL, tamoxifen-d 5500 ng/mL, capecitabine-d 111000ng/mL and crizotinib-d 52000 ng/mL
The serum mentioned in the invention is human or animal serum.
In a preferred embodiment, a kit for detecting the concentration of 11 anti-tumor drugs in serum comprises the following reagents:
(1) eluent:
eluent A: 0.1% formic acid, 2mM ammonium acetate in water; eluent B: acetonitrile;
(2) calibration solution:
the following concentrations of the master solutions were prepared: the above standards were formulated at the following concentrations: afatinib 0.1mg/mL, apatinib 0.1mg/mL, gefitinib 5mg/mL, erlotinib hydrochloride 2mg/mL, crizotinib 0.1mg/mL, imatinib 2mg/mL, sunitinib 0.1mg/mL, N-dethylsunitinib 0.1mg/mL, erlotinib 2mg/mL, tamoxifen 2mg/mL and capecitabine 2 mg/mL;
transferring a standard product mother solution: afatinib 20 μ L, apatinib 40 μ L, gefitinib 4 μ L, erlotinib hydrochloride 5 μ L, crizotinib 50 μ L, imatinib 10 μ L, sunitinib 20 μ L, N-desethylsunitinib 20 μ L, erlotinib 20 μ L, tamoxifen 10 μ L, and capecitabine 5 μ L; then adding into 296 μ L50% methanol water to obtain 500 μ L standard stock solution; the concentrations are given in table 1 below.
TABLE 1 stock solutions of standards
The invention prepares the stock solution of the mixed standard substance into the solutions of the calibrator with seven different concentration points by using a blank serum substrate (blank serum without target drugs), and the preparation process is as follows:
adding 10 mu L of standard stock solution into 190 mu L of blank serum matrix to serve as a first high-value concentration point; diluting 50 μ L of the first high-value concentration point with 50 μ L of blank serum matrix to obtain a second high-value concentration point; diluting the first high-value concentration point with 9 times volume of blank serum substrate to obtain a third high-value concentration point; diluting the second high-value concentration point with 9 times volume of blank serum substrate to obtain a fourth high-value concentration point; diluting the third high-value concentration point with 9 times volume of blank serum substrate to obtain a fifth high-value concentration point; diluting the fourth high-value concentration point with 9 times volume of blank serum matrix to obtain a sixth high-value concentration point; and (4) taking the fifth high-value concentration point, and diluting the fifth high-value concentration point with 5 times of volume of blank serum substrate to obtain a seventh high-value concentration point.
The invention adopts a gradient dilution method to prepare standard yeast, after a standard solution is taken out from a refrigerator at minus 80 ℃, the standard solution is vortexed for 10s, the maximum concentration point of the standard yeast is prepared by the standard solution within 2min, and the standard yeast is stored at minus 80 ℃ after the preparation, and the specific process is as shown in the following table 2 (unit: ng/mL).
TABLE 2 Standard koji preparation
| Standard song | Pipetting solution (mu L) | Blank serum matrix (μ L) |
| S7 | Mixed |
190 |
| S6 | S7 50 | 50 |
| S5 | S7 20 | 180 |
| S4 | S6 20 | 180 |
| S3 | S5 20 | 180 |
| S2 | S4 20 | 180 |
| S1 | S3 25 | 125 |
(3) Internal standard solution:
the following internal standard mother liquor was prepared: afatinib-d 61 mg/mL, apatinib-d 81 mg/mL, gefitinib-d 31mg/mL, imatinib-d 81 mg/mL, sunitinib-d 40.1mg/mL, N-desethylsunitinib-d 50.1mg/mL, tamoxifen-d 50.1mg/mL, capecitabine-d 110.1mg/mL and crizotinib-d 51 mg/mL;
transferring internal standard mother liquor: afatinib-d 62.5 μ L, apatinib-d 81 μ L, gefitinib-d 31 μ L, imatinib-d 82.5 μ L, sunitinib-d 42 μ L, N-desethylsunitinib-d 52 μ L, tamoxifen-d 52.5 μ L, capecitabine-d 115 μ L, and crizotinib-d 51 μ L; then adding into 480.5 μ L50% methanol water to obtain 500 μ L internal standard solution; the concentrations refer to table 3 below.
TABLE 3 internal standard solution preparation
(4) Protein precipitant:
a mixed solution of methanol and acetonitrile, wherein the volume ratio of the methanol to the acetonitrile is 1: 2;
(5) quality control product:
the mixed standard stock solution is prepared into QC (L), QC (M) and QC (H) with three different concentrations by using blank serum without target drugs, wherein the three different concentrations are specifically shown in table 4.
TABLE 4 corresponding concentration of quality control (unit ng/mL)
| Compound ID | Compound (I) | QC(L) | QC(M) | QC(H) |
| 1 | AFT | 0.8 | 8 | 80 |
| 2 | APT | 1.6 | 16 | 160 |
| 3 | |
8 | 80 | 800 |
| 4 | |
4 | 40 | 400 |
| 5 | |
2 | 20 | 200 |
| 6 | |
8 | 80 | 800 |
| 7 | SNT | 0.8 | 8 | 80 |
| 8 | NSNT | 0.8 | 8 | 80 |
| 9 | ELT | 16 | 160 | 1600 |
| 10 | |
8 | 80 | 800 |
| 11 | |
4 | 40 | 400 |
QC (L) includes: afatinib 0.8ng/mL, Apatinib 1.6ng/mL, gefitinib 8ng/mL, hydrochloric acid of Icotinib 4ng/mL, crizotinib 2ng/mL, imatinib 8ng/mL, sunitinib 0.8ng/mL, N-ethyl sunitinib 0.8ng/mL, erlotinib 16ng/mL, tamoxifen 8ng/mL, capecitabine 4 ng/mL.
QC (M) comprises: afatinib 8ng/mL, Apatinib 16ng/mL, gefitinib 80ng/mL, hydrochloric acid of Icotinib 40ng/mL, crizotinib 20ng/mL, imatinib 80ng/mL, sunitinib 8ng/mL, N-ethyl sunitinib 8ng/mL, erlotinib 160ng/mL, tamoxifen 80ng/mL, capecitabine 40 ng/mL.
QC (H) includes: afatinib 80ng/mL, Apatinib 160ng/mL, gefitinib 800ng/mL, hydrochloric acid of Icotinib 400ng/mL, crizotinib 200ng/mL, imatinib 800ng/mL, sunitinib 80ng/mL, N-ethyl sunitinib 80ng/mL, erlotinib 1600ng/mL, tamoxifen 800ng/mL, capecitabine 400 ng/mL.
When the kit is used for detecting the concentration of 11 anti-tumor drugs in serum, an internal standard solution and a protein precipitator are mixed to prepare the protein precipitator containing the internal standard. In a preferable scheme, the volume ratio of the internal standard solution to the protein precipitator is 0.1-0.3: 19.9-19.7. In a more preferred embodiment, the volume ratio of the internal standard solution to the protein precipitant is 0.02:19.98(1: 999). For example, the protein precipitant containing the internal standard is prepared by mixing an internal standard solution and a protein precipitant (methanol and acetonitrile in a volume ratio of 1:2) in a volume ratio of 1: 999.
The application of the kit in detecting the concentrations of the 11 anti-tumor drugs in the serum by using the ultra-performance liquid chromatography tandem mass spectrometry technology is also within the protection scope of the invention.
The specific detection method comprises the following steps:
a method for detecting the concentration of 11 anti-tumor drugs in serum,
the 11 anti-tumor drugs are: capecitabine (CPT), Afatinib (AFT), Tamoxifen (TMX), Gefitinib (GFT), Imatinib (IMT), Sunitinib (SNT) Erlotinib (ELT), N-desethylsunitinib (NSNT), erlotinib hydrochloride (ICT), Apatinib (APT) and Crizotinib (CZP);
the internal standard substances corresponding to the 11 antitumor drugs are as follows: capecitabine-d 11(CPT-d11), Afatinib-d 6(AFT-d6), tamoxifen-d 5(TMX-d5), gefitinib-d 3(GFT-d3), imatinib-d 8(IMT-d8), sunitinib-d 4(SNT-d4), N-desethylsunitinib-d 5(NSNT-d5), apatinib-d 8(APT-d8), and crizotinib-d 5(CZP-d 5).
After a serum sample is pretreated, the serum sample is oscillated and centrifuged, supernatant is taken for sample injection, 11 antitumor drugs in the pretreated serum are detected by adopting an ultra high performance liquid chromatography tandem mass spectrometry technology, an object to be detected is separated from a serum matrix by using the ultra high performance liquid chromatography, then a mass spectrum internal standard quantitative method is used, the concentration ratio of a standard substance to an internal standard substance is taken as an X axis, the peak area ratio of the standard substance to the internal standard substance is taken as a Y axis, a calibration curve is established, and the content of the 11 antitumor drugs is calculated.
The specific chromatographic conditions are as follows:
(1) ultra-high performance liquid chromatography conditions:
mobile phase A: 0.01 to 0.2 percent of formic acid and 1 to 3mM of ammonium acetate aqueous solution; mobile phase B: acetonitrile;
the type of the chromatographic column: phenomenex Kinetex XB-C18;
a mixed mobile phase A and a mixed mobile phase B are adopted for gradient elution, and the initial ratio of the mobile phase A to the mobile phase B is 85-100: 25-0; the gradient elution procedure was as follows: in 0-1.0 min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 85-100: 25-0 to 70:30 at a constant speed; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 70:30 to 2:98 at a constant speed within 1.0-3.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is kept at 2:98 within 3.0-4.0 minutes, the volume ratio of the mobile phase A to the mobile phase B is changed from 2:98 to the initial ratio within 4.0-6.5 minutes, and the collection time of each sample is 6.5 minutes;
(2) mass spectrum conditions:
in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 3.0kV (ESI +); the temperature of the drying gas is 300 ℃; the pressure of atomizing gas is 45psi, the temperature of sheath gas is 350 ℃, and the flow rate of the sheath gas is 11L/min; each target and its corresponding internal standard were monitored simultaneously.
In order to improve the chromatographic separation selectivity, it may be considered to adjust the polarity of the mobile phase. The invention adds formic acid and ammonium acetate into the mobile phase A, can effectively improve the ionization efficiency of the target compound, has higher sensitivity than the prior art which adopts an LC-MS/MS method to detect 11 anti-tumor drugs in serum under the coordination of other conditions, has simple pretreatment process, low cost, high sensitivity and strong specificity, and completes the separation and detection of the 11 anti-tumor drugs within 6.5 minutes. In a preferred embodiment, the mobile phase A is 0.01% -0.2% formic acid, 1 mM-3 mM ammonium acetate aqueous solution, and preferably, the mobile phase A is 0.1% formic acid, 2mM ammonium acetate aqueous solution without affecting the effect of the present invention.
In chromatography, the choice of the chromatographic column is important and the requirements for the chromatographic column: high column efficiency, good selectivity, high analysis speed and the like. Acetonitrile, 0.01-0.2% formic acid and 1-3 mM ammonium acetate aqueous solution are used as mobile phases, the type of a chromatographic column is Phenomenex Kinetex XB-C18 (3.0X 50mM,2.6 mu m), endogenous substances do not interfere with the determination of a sample under the coordination of other conditions, the sensitivity is high, the specificity is strong, and the accuracy and the precision basically meet the requirements.
When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be capable of being added to the sample in an accurate, known amount, and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. The isotope is used as the internal standard, the internal standard and the substance to be detected have the same chemical property and matrix effect, and the repeatability and the accuracy are better when the concentration of the 11 anti-tumor drugs in serum is measured.
In a preferable scheme, the initial ratio of the mobile phase A to the mobile phase B is 85-100: 25-0. Further preferably, the initial ratio of mobile phase a and mobile phase B is 99: 1.
In a preferred embodiment, the flow rate is 0.2-0.5 mL/min, preferably 0.3 mL/min.
Further, the column temperature is 40-50 ℃, preferably 45 ℃.
In one embodiment, the injection volume is 0.2-2 μ L, preferably 1 μ L.
In a preferred scheme, when the ultra performance liquid chromatography tandem mass spectrometry technology is adopted to detect 11 antitumor drugs in pretreated serum, the specific chromatographic conditions are as follows:
(1) ultra-high performance liquid chromatography conditions:
mobile phase A: 0.1% formic acid, 2mM ammonium acetate in water; mobile phase B: acetonitrile;
the type of the chromatographic column: phenomenex Kinetex XB-C18 (3.0X 50mm,2.6 μm);
adopting a mode of gradient elution by taking a mobile phase A and a mobile phase B as a mixed mobile phase, wherein the initial ratio of the mobile phase A to the mobile phase B is 99: 1; the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 99:1 to 70:30 at a constant speed within 0-1.0 min; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 70:30 to 2:98 at a constant speed within 1.0-3.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is kept at 2:98 within 3.0-4.0 minutes, the volume ratio of the mobile phase A to the mobile phase B is changed from 2:98 to the initial ratio within 4.0-6.5 minutes, and the collection time of each sample is 6.5 minutes; the gradient elution mode is specifically shown in table 1; the flow rate was 0.3mL/min, the column temperature was 45 ℃ and the injection volume was 1. mu.L.
TABLE 5 mobile phase gradient elution parameters
| Time (min) | Flow rate (mL/min) | ％A | ％B | Curve |
| 0.0 | 0.3 | 99 | 1 | - |
| 1.0 | 0.3 | 70 | 30 | 6 |
| 3.0 | 0.3 | 2 | 98 | 6 |
| 4.0 | 0.3 | 2 | 98 | 6 |
| 6.5 | 0.3 | 99 | 1 | 1 |
(2) Mass spectrum conditions:
in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 3.0kV (ESI +); the temperature of the drying gas is 300 ℃; the pressure of atomizing gas is 45psi, the temperature of sheath gas is 350 ℃, and the flow rate of the sheath gas is 11L/min; simultaneously monitoring each target and the corresponding internal standard thereof; the mass spectrum source parameters are shown in table 6, and the mass spectrum parameters of each target and the corresponding isotope internal standard thereof are simultaneously monitored and shown in table 7.
TABLE 6 Mass Spectrometry Source parameters
| Item | Parameter(s) |
| Capillary voltage (kV) | 3.0 |
| Temperature of drying gas (. degree.C.) | 300 |
| Atomizer pressure (psi) | 45 |
| Temperature of sheath gas (. degree. C.) | 350 |
| Sheath gas flow rate (L/min) | 11 |
TABLE 711 parameters of Mass Spectrometry for antitumor drug detection
The serum mentioned in the invention is human or animal serum.
In a preferred embodiment, the pretreated serum of the present invention is prepared as follows: putting 50 mu L of serum into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitator containing internal standard work into the centrifuge tube, oscillating for 3-10 min, centrifuging for 4-10 min at 12000-15000 r/min and 4-20 ℃, taking supernatant into a sample injection bottle, and performing sample injection; the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 2.
In a preferred embodiment, the pretreated serum of the present invention is prepared as follows: putting 50 mu L of serum into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitator containing internal standard work into the centrifuge tube, oscillating for 3-10 min, centrifuging for 4-10 min at 12000-15000 r/min and 4-20 ℃, taking supernatant into a sample injection bottle, and performing sample injection; the protein precipitator containing the internal standard is prepared by mixing an internal standard solution and a protein precipitator, wherein the volume ratio of the internal standard solution to the protein precipitator is 0.1-0.3: 19.9-19.7.
In a more preferred embodiment, the pretreated serum of the present invention is prepared as follows: 50 mu L of serum is taken to be put into a 1.5mL centrifuge tube, 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:2) is added into the centrifuge tube, the mixture is shaken for 5min, and after the centrifuge tube is centrifuged for 5min at 14000r/min and 15 ℃, 60 mu L of supernatant is taken to be put into a sample injection bottle and injected for 1 mu L, wherein the protein precipitant containing the internal standard is prepared by mixing an internal standard solution and the protein precipitant, and the volume ratio of the internal standard solution to the protein precipitant is 0.02:19.98 (namely 1: 999).
In one embodiment, the protein precipitant containing the internal standard is prepared as follows:
the following internal standard mother liquor was prepared: afatinib-d 61 mg/mL, apatinib-d 81 mg/mL, gefitinib-d 31mg/mL, imatinib-d 81 mg/mL, sunitinib-d 40.1mg/mL, N-desethylsunitinib-d 50.1mg/mL, tamoxifen-d 50.1mg/mL, capecitabine-d 110.1mg/mL and crizotinib-d 51 mg/mL;
transferring internal standard mother liquor: afatinib-d 62.5 μ L, apatinib-d 81 μ L, gefitinib-d 31 μ L, imatinib-d 82.5 μ L, sunitinib-d 42 μ L, N-desethylsunitinib-d 52 μ L, tamoxifen-d 52.5 μ L, capecitabine-d 115 μ L, and crizotinib-d 51 μ L; then adding into 480.5 μ L methanol water to obtain 500 μ L standard stock solution;
and adding 20 mu L of the internal standard solution into 19.98mL of protein precipitator to obtain the protein precipitator containing the internal standard.
In one scheme, the protein precipitator is a mixed solution of methanol and acetonitrile; preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 1-3. More preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 2.
In one embodiment, the standard solution is prepared as follows:
preparing a standard mother solution with the following concentration: afatinib 0.1mg/mL, apatinib 0.1mg/mL, gefitinib 5mg/mL, erlotinib hydrochloride 2mg/mL, crizotinib 0.1mg/mL, imatinib 2mg/mL, sunitinib 0.1mg/mL, N-dethylsunitinib 0.1mg/mL, erlotinib 2mg/mL, tamoxifen 2mg/mL and capecitabine 2 mg/mL;
transferring a standard product mother solution: afatinib 20 μ L, apatinib 40 μ L, gefitinib 4 μ L, erlotinib hydrochloride 5 μ L, crizotinib 50 μ L, imatinib 10 μ L, sunitinib 20 μ L, N-desethylsunitinib 20 μ L, erlotinib 20 μ L, tamoxifen 10 μ L, and capecitabine 5 μ L; add to 296. mu.L of methanol water to give 500. mu.L of standard stock solution. The standard stock solution comprises: afatinib 4 μ g/mL, Apatinib 8 μ g/mL, gefitinib 40 μ g/mL, hydrochloric acid of Icotinib 20 μ g/mL, crizotinib 10 μ g/mL, imatinib 40 μ g/mL, sunitinib 4 μ g/mL, N-ethyl sunitinib 4 μ g/mL, erlotinib 80 μ g/mL, tamoxifen 40 μ g/mL and capecitabine 20 μ g/mL.
Preparing the standard stock solution into calibration solution with seven different concentration points by using a blank serum substrate, wherein the seven concentration points of the calibration solution are as follows:
the seven concentration points of afatinib, sunitinib and N-dethylsunitinib are as follows in sequence: 0.4ng/mL, 1ng/mL, 2ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 200 ng/mL;
the seven concentration points of apatinib are in sequence: 0.8ng/mL, 2ng/mL, 4ng/mL, 20ng/mL, 40ng/mL, 200ng/mL, 400 ng/mL;
seven concentration points of crizotinib are in order: 1ng/mL, 2.5ng/mL, 5ng/mL, 25ng/mL, 50ng/mL, 250ng/mL, 500 ng/mL;
the seven concentration points of the icotinib hydrochloride and capecitabine are as follows: 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000 ng/mL;
seven concentration points of gefitinib, imatinib and tamoxifen are as follows: 4ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000 ng/mL;
seven concentration points of erlotinib are in order: 8ng/mL, 20ng/mL, 40ng/mL, 200ng/mL, 400ng/mL, 2000ng/mL, 4000 ng/mL.
50 mu L of sample at each concentration point is taken and put into a 1.5mL centrifuge tube, 200 mu L of protein precipitant containing internal standard (the volume ratio of methanol to acetonitrile is 1:2) is added into the centrifuge tube, the mixture is shaken for 5min, and after the mixture is centrifuged for 5min at 14000r/min and 15 ℃, 60 mu L of supernatant is taken and put into an injection bottle, and 1uL of sample is injected.
The invention also comprises a quality control product prepared by the following method: preparing the mixed standard stock solution into QC (L), QC (M) and QC (H) with three different concentrations by using blank serum without target drugs, wherein,
qc (l) was a 5000-fold dilution of the above standard stock solution in blank serum matrix.
Qc (m) is a 500-fold dilution of the above standard stock solution in blank serum matrix.
Qc (h) was diluted 50-fold with blank serum matrix for the standard stock solution described above.
QC (L) includes: afatinib 0.8ng/mL, Apatinib 1.6ng/mL, gefitinib 8ng/mL, hydrochloric acid of Icotinib 4ng/mL, crizotinib 2ng/mL, imatinib 8ng/mL, sunitinib 0.8ng/mL, N-ethyl sunitinib 0.8ng/mL, erlotinib 16ng/mL, tamoxifen 8ng/mL, capecitabine 4 ng/mL.
QC (M) comprises: afatinib 8ng/mL, Apatinib 16ng/mL, gefitinib 80ng/mL, hydrochloric acid of Icotinib 40ng/mL, crizotinib 20ng/mL, imatinib 80ng/mL, sunitinib 8ng/mL, N-ethyl sunitinib 8ng/mL, erlotinib 160ng/mL, tamoxifen 80ng/mL, capecitabine 40 ng/mL.
QC (H) includes: afatinib 80ng/mL, Apatinib 160ng/mL, gefitinib 800ng/mL, hydrochloric acid of Icotinib 400ng/mL, crizotinib 200ng/mL, imatinib 800ng/mL, sunitinib 80ng/mL, N-ethyl sunitinib 80ng/mL, erlotinib 1600ng/mL, tamoxifen 800ng/mL, capecitabine 400 ng/mL.
The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.
Example 1
First, experimental material and instrument
1. Material
The samples were obtained from serum samples collected from the outpatient clinic of 2019 months in the Nanjing drugstore Hospital.
(1) The instrument comprises the following steps: qlife Lab 9000plus triple quadrupole mass spectrometer (department of health); qlife Lab 9000 ultra performance liquid chromatography system (with G7167A autosampler, department of pediatrics); SCILOGEX D2012 high speed bench top centrifuge (usa); ultra pure water meter (ELGA LabWater, uk); multi-tube Vortex mixer (Vortex genie2, usa); an adjustable pipettor (Eppendorf 0.5-10 muL, 10-100 muL, 100-1000 muL); glassware, graduated cylinders, and the like.
(2) Reagent consumables: MS grade acetonitrile (Fisher, usa); HPLC grade methanol (Honeywell, usa); HPLC grade acetonitrile (Honeywell, usa); MS grade formic acid (Fisher, usa); MS grade ammonium acetate (Sigma, usa) column model: phenomenex Kinetex XB-C18 (3.0X 50mm,2.6 μm).
(3) And (3) standard substance: the standards and their corresponding internal standards are shown in table 8 below.
TABLE 8 Standard and internal standards
(4) Quality control product: blank sera containing 11 antitumor drugs were divided into low, medium and high concentrations, qc (l), qc (m) and qc (h), as shown in table 4.
The upper and lower peripheries of the kit are coated with films, the kit is shockproof and heat-insulated, mobile phases A and B are placed on the upper left, and 11 ampoules are respectively placed on the lower left, wherein the standard solution and the quality control product are respectively; 50mL of protein precipitant was placed on the right side.
Second, liquid condition
(1) Chromatographic conditions are as follows: chromatographic conditions are as follows: mobile phase A: 0.1% formic acid, 2mM ammonium acetate in water; mobile phase B: and (3) acetonitrile. The type of the chromatographic column: phenomenex Kinetex XB-C18 (3.0X 50mm,2.6 μm) using gradient elution, as detailed in Table 5. The flow rate was 0.3mL/min, the column temperature was 45 ℃ and the injection volume was 1. mu.L.
(2) In an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 3.0kV (ESI +); the temperature of the drying gas is 300 ℃; the pressure of atomizing gas is 45psi, the temperature of sheath gas is 350 ℃, and the flow rate of the sheath gas is 11L/min; the mass spectrometry source parameters are shown in table 6, while monitoring each target and its corresponding internal standard, the mass spectrometry parameters of each target are shown in table 7.
Third, the experimental process
(1) Preparing a standard substance:
the 11 antitumor drugs are prepared into standard mother liquor with the following concentrations: afatinib 0.1mg/mL, apatinib 0.1mg/mL, gefitinib 5mg/mL, erlotinib hydrochloride 2mg/mL, crizotinib 0.1mg/mL, imatinib 2mg/mL, sunitinib 0.1mg/mL, N-dethylsunitinib 0.1mg/mL, erlotinib 2mg/mL, tamoxifen 2mg/mL and capecitabine 2 mg/mL;
transferring a standard product mother solution: afatinib 20 μ L, apatinib 40 μ L, gefitinib 4 μ L, erlotinib hydrochloride 5 μ L, crizotinib 50 μ L, imatinib 10 μ L, sunitinib 20 μ L, N-desethylsunitinib 20 μ L, erlotinib 20 μ L, tamoxifen 10 μ L, and capecitabine 5 μ L; then added to 296. mu.L of 50% methanol water to give 500. mu.L of a standard stock solution. See table 1 for details.
The standard stock solution is prepared into a calibrator solution with seven different concentration points by using a blank serum matrix (blank serum without a target drug), which is detailed in table 2, wherein the seven concentration points of the calibrator solution are as follows:
the seven concentration points of afatinib, sunitinib and N-dethylsunitinib are as follows in sequence: 0.4ng/mL, 1ng/mL, 2ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 200 ng/mL;
the seven concentration points of apatinib are in sequence: 0.8ng/mL, 2ng/mL, 4ng/mL, 20ng/mL, 40ng/mL, 200ng/mL, 400 ng/mL;
seven concentration points of crizotinib are in order: 1ng/mL, 2.5ng/mL, 5ng/mL, 25ng/mL, 50ng/mL, 250ng/mL, 500 ng/mL;
the seven concentration points of the icotinib hydrochloride and capecitabine are as follows: 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000 ng/mL;
seven concentration points of gefitinib, imatinib and tamoxifen are as follows: 4ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000 ng/mL;
seven concentration points of erlotinib are in order: 8ng/mL, 20ng/mL, 40ng/mL, 200ng/mL, 400ng/mL, 2000ng/mL, 4000 ng/mL.
(2) Preparation of internal standard solution
The following internal standard mother liquor was prepared: afatinib-d 61 mg/mL, apatinib-d 81 mg/mL, gefitinib-d 31mg/mL, imatinib-d 81 mg/mL, sunitinib-d 40.1 mg/mL, N-desethylsunitinib-d 50.1mg/mL, tamoxifen-d 50.1mg/mL, capecitabine-d 110.1 mg/mL, and crizotinib-d 51 mg/mL.
Transferring internal standard mother liquor: afatinib-d 62.5 μ L, apatinib-d 81 μ L, gefitinib-d 31 μ L, imatinib-d 82.5 μ L, sunitinib-d 42 μ L, N-desethylsunitinib-d 52 μ L, tamoxifen-d 52.5 μ L, capecitabine-d 115 μ L, and crizotinib-d 51 μ L; then 480.5 μ L. A500. mu.L standard stock solution was obtained with Afatinib-d 65000ng/mL, Afatinib-d 82000 ng/mL, gefitinib-d 32000 ng/mL, imatinib-d 85000 ng/mL, sunitinib-d 4400 ng/mL, N-desethylsunitinib-d 5400 ng/mL, tamoxifen-d 5500 ng/mL, capecitabine-d 111000ng/mL, and crizotinib-d 52000 ng/mL. See table 3 for details.
(3) Preparing a quality control product:
the standard stock solution was taken and prepared into three different concentrations of QC (L), QC (M) and QC (H) with blank serum without target drug, as shown in Table 4.
QC (L) includes: afatinib 0.8ng/mL, Apatinib 1.6ng/mL, gefitinib 8ng/mL, hydrochloric acid of Icotinib 4ng/mL, crizotinib 2ng/mL, imatinib 8ng/mL, sunitinib 0.8ng/mL, N-ethyl sunitinib 0.8ng/mL, erlotinib 16ng/mL, tamoxifen 8ng/mL, capecitabine 4 ng/mL.
QC (M) comprises: afatinib 8ng/mL, Apatinib 16ng/mL, gefitinib 80ng/mL, hydrochloric acid of Icotinib 40ng/mL, crizotinib 20ng/mL, imatinib 80ng/mL, sunitinib 8ng/mL, N-ethyl sunitinib 8ng/mL, erlotinib 160ng/mL, tamoxifen 80ng/mL, capecitabine 40 ng/mL.
QC (H) includes: afatinib 80ng/mL, Apatinib 160ng/mL, gefitinib 800ng/mL, hydrochloric acid of Icotinib 400ng/mL, crizotinib 200ng/mL, imatinib 800ng/mL, sunitinib 80ng/mL, N-ethyl sunitinib 80ng/mL, erlotinib 1600ng/mL, tamoxifen 800ng/mL, capecitabine 400 ng/mL.
(4) Sample processing
1) Treating a standard substance: taking 50 mu L of each concentration point of seven different calibrator samples, putting the sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:2), oscillating for 5min, centrifuging for 5min at 14000r/min and 15 ℃, taking 60 mu L of supernatant into an injection bottle, and injecting 1 mu L of supernatant.
2) Pretreatment of a serum sample: 50 mu L of the supernatant is put into a 1.5mL centrifuge tube, 200 mu L of protein precipitant containing internal standard (the volume ratio of methanol to acetonitrile is 1:2) is added into the centrifuge tube, the mixture is shaken for 5min, and after the centrifuge tube is centrifuged for 5min at 14000r/min and 15 ℃, 60 mu L of the supernatant is taken into a sample injection bottle and injected into 1 mu L.
3) Pretreatment of quality control products: the quality control solutions QC (L), QC (M), QC (H) are respectively taken and 50 μ L of each quality control solution QC (L), QC (M), QC (H) are respectively put into a 1.5mL centrifuge tube, and then the quality control solutions QC (L), QC (M), QC (H) are consistent with the pretreatment of the serum sample, and the details are not.
The components of the assay kit are shown in Table 9.
Preparation of kit components for analyzing concentrations of 911 antitumor drugs in table
Remarking: the protein precipitant containing the internal standard is prepared by the following method, and 20 mu L of the internal standard solution is added into 19.98mL of the protein precipitant to obtain the protein precipitant containing the internal standard.
Fourth, method verification
1. Extracting an ion current chromatogram: the peak shapes of the 11 antitumor drug standards and the serum sample are symmetrical, and there is no peak interference, which indicates that good detection can be obtained under the conditions, and fig. 1 is an extracted ion flow chart of the 11 antitumor drug standards; FIG. 2 is an ion flow chart of 11 antitumor drugs extracted from serum samples.
2. Calibration curve: and establishing a calibration curve by adopting an internal standard quantitative method and utilizing TargetLynx software to calculate the concentration of the substance to be detected in the serum by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis. The linear fitting equation of the 11 antitumor drugs in the respective concentration ranges has good linearity, the correlation coefficient is more than 0.99, and the quantitative requirements are met, which is shown in Table 10.
TABLE 1011 linear regression equation and linear correlation coefficient for antitumor drugs
3. Accuracy survey: and evaluating the accuracy of the method by adopting a standard recovery rate test. A mixed blank serum sample is prepared, 3 concentrations of mixed standard substances of low, medium and high are respectively added, the same steps are repeated for 5 times of treatment and measurement, the result shows that the standard addition recovery rate of 11 antitumor drugs is between 96.16% and 107.13%, the RSD of 5 repeated tests is in the range of 1.45% to 6.68%, and the statistical result is shown in Table 11.
Results of recovery of the addition of the top 1111 antitumor drugs
4. And (3) precision test: taking an interference-free blank serum sample, adding 11 anti-tumor drug standards with different concentrations to obtain serum samples with low, medium and high concentrations, repeatedly processing 6 batches in one day for three days continuously, quantitatively determining the concentrations of the 11 anti-tumor drugs by an internal standard method, carrying out batch processing with the internal precision of 2.34-13.90 percent and carrying out batch processing with 3 batches in three days, wherein the inter-batch precision is calculated to be 0.38-7.78 percent, and the results are shown in Table 12.
TABLE 12 results of the Intra-and Inter-batch precision measurements
By adopting the kit, the concentration of the 11 anti-tumor drugs in the human serum can be simultaneously determined by an ultra-high performance liquid chromatography-tandem mass spectrometry (ID-HPLC-MS/MS) technology, the serum dosage is less (only 50 mu L), the pretreatment is simple, and the analysis of various substances by one needle only needs 6.5 minutes, so that the kit is simple and rapid.
The internal standard method is used for quantification, so that the matrix interference can be greatly eliminated, the result is not influenced by conditions such as a pretreatment process and instrument response fluctuation, and accurate quantification can be achieved. The result of the accuracy of the method is evaluated by the standard recovery test, and shows that the standard recovery of the 11 anti-tumor drugs is between 90.63% and 107.13%, the RSD of 5 times of repeated tests is in the range of 1.45% to 8.55%, and the accuracy is good.
The reproducibility result of the method shows that the intra-batch precision of the 11 antitumor drugs is 2.34-13.90%, the inter-batch precision is 0.38-13.49%, and the reproducibility of the method is good. The pre-treatment process of the established serum sample is very simple, and the serum dosage is only 50 mu L.
In a word, the kit provided by the invention has the advantages of high sensitivity, strong specificity, accuracy and simple pretreatment process, the separation and detection of the compounds can be completed within 6.5 minutes, the accuracy and precision meet the requirements, and the kit can be used for quantitative analysis of the concentration of 11 anti-tumor drugs in serum clinically and provides a reliable detection method for monitoring the concentration of related drugs.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A kit for detecting the concentration of 11 antitumor drugs in serum is characterized in that: the kit comprises: eluent, calibrator solution, internal standard solution, protein precipitator and quality control product;
the 11 anti-tumor drugs are: capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib;
the eluent comprises an eluent A and an eluent B, wherein the eluent A is a mixed solution of 0.01% -0.2% formic acid and 1 mM-3 mM ammonium acetate water solution, and the eluent B is acetonitrile;
the calibrator solution comprises a mixed solution of a series of known concentrations of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib prepared from a blank serum matrix;
the internal standard solution is a methanol water solution containing Afatinib-d 6, apatinib-d 8, gefitinib-d 3, imatinib-d 8, sunitinib-d 4, N-dethylsunitinib-d 5, tamoxifen-d 5, capecitabine-d 11 and crizotinib-d 5;
the protein precipitator is a mixed solution of methanol and acetonitrile;
the quality control product is a mixed solution of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-dethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib which are prepared from a blank serum matrix and have known concentration.
2. The kit of claim 1, wherein: the eluent A is a mixed solution of 0.1% formic acid and 2mM ammonium acetate aqueous solution.
3. The kit of claim 1, wherein: the calibrator solution is a mixed solution of the following 7 solutions:
mixed solution 1: the concentrations of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib are respectively: capecitabine 2ng/mL, Afatinib 0.4ng/mL, tamoxifen 4ng/mL, gefitinib 4ng/mL, imatinib 4ng/mL, sunitinib 0.4ng/mL, erlotinib 8ng/mL, N-dethylsunitinib 0.4ng/mL, erlotinib hydrochloride 2ng/mL, apatinib 0.8ng/mL, crizotinib 1 ng/mL;
mixed solution 2: the concentrations of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib are respectively: capecitabine 5ng/mL, Afatinib 1ng/mL, tamoxifen 10ng/mL, gefitinib 10ng/mL, imatinib 10ng/mL, sunitinib 1ng/mL, erlotinib 20ng/mL, N-dethylsunitinib 1ng/mL, erlotinib hydrochloride 5ng/mL, Apatinib 0.8ng/mL, crizotinib 2.5 ng/mL;
mixed solution 3: the concentrations of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib are respectively: capecitabine 10ng/mL, Afatinib 2ng/mL, tamoxifen 20ng/mL, gefitinib 20ng/mL, imatinib 20ng/mL, sunitinib 2ng/mL, erlotinib 40ng/mL, N-dethylsunitinib 2ng/mL, erlotinib hydrochloride 10ng/mL, Apatinib 4ng/mL, crizotinib 5 ng/mL;
mixed solution 4: the concentrations of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib are respectively: 50ng/mL of capecitabine, 10ng/mL of Afatinib, 100ng/mL of tamoxifen, 100ng/mL of gefitinib, 100ng/mL of imatinib, 10ng/mL of sunitinib, 200ng/mL of erlotinib, 10ng/mL of N-desethylsunitinib, 50ng/mL of Icotinib hydrochloride, 20ng/mL of Apatinib and 25ng/mL of crizotinib;
mixed solution 5: the concentrations of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib are respectively: capecitabine 100ng/mL, Afatinib 20ng/mL, tamoxifen 200ng/mL, gefitinib 200ng/mL, imatinib 200ng/mL, sunitinib 20ng/mL, erlotinib 400ng/mL, N-desethylsunitinib 20ng/mL, erlotinib hydrochloride 100ng/mL, Apatinib 40ng/mL, crizotinib 50 ng/mL;
mixed solution 6: the concentrations of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib are respectively: capecitabine 500ng/mL, Afatinib 100ng/mL, tamoxifen 1000ng/mL, gefitinib 1000ng/mL, imatinib 1000ng/mL, sunitinib 100ng/mL, erlotinib 2000ng/mL, N-dethylsunitinib 100ng/mL, erlotinib hydrochloride 500ng/mL, Apatinib 200ng/mL, crizotinib 250 ng/mL;
mixed solution 7: the concentrations of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib are respectively: capecitabine 1000ng/mL, Afatinib 200ng/mL, tamoxifen 2000ng/mL, gefitinib 2000ng/mL, imatinib 2000ng/mL, sunitinib 200ng/mL, erlotinib 4000ng/mL, N-desethylsunitinib 200ng/mL, erlotinib hydrochloride 1000ng/mL, Apatinib 400ng/mL, crizotinib 500 ng/mL.
4. The kit of claim 1, wherein: the internal standard solution is a methanol aqueous solution containing Afatinib-d 65 mu g/mL, Apatinib-d 82 mu g/mL, gefitinib-d 32 mu g/mL, imatinib-d 85 mu g/mL, sunitinib-d 40.4 mu g/mL, N-dethylsunitinib-d 50.4 mu g/mL, tamoxifen-d 50.5 mu g/mL, capecitabine-d 111 mu g/mL and crizotinib-d 52 mu g/mL.
5. The kit of claim 4, wherein: the methanol aqueous solution is 40% -60% v/v methanol aqueous solution.
6. The kit of claim 1, wherein: the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 1-3.
7. The kit of claim 1, wherein: the quality control product is 3 mixed solutions as follows:
low concentration quality control product: the concentrations of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib are respectively: afatinib 0.8ng/mL, Apatinib 1.6ng/mL, gefitinib 8ng/mL, hydrochloric acid of Icotinib 4ng/mL, crizotinib 2ng/mL, imatinib 8ng/mL, sunitinib 0.8ng/mL, N-ethyl sunitinib 0.8ng/mL, erlotinib 16ng/mL, tamoxifen 8ng/mL, capecitabine 4 ng/mL;
medium concentration quality control: the concentrations of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib are respectively: afatinib 8ng/mL, Apatinib 16ng/mL, gefitinib 80ng/mL, hydrochloric acid of Icotinib 40ng/mL, crizotinib 20ng/mL, imatinib 80ng/mL, sunitinib 8ng/mL, N-ethyl sunitinib 8ng/mL, erlotinib 160ng/mL, tamoxifen 80ng/mL, capecitabine 40 ng/mL;
high concentration quality control product: the concentrations of capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethylsunitinib, erlotinib hydrochloride, apatinib and crizotinib are respectively: afatinib 80ng/mL, Apatinib 160ng/mL, gefitinib 800ng/mL, hydrochloric acid of Icotinib 400ng/mL, crizotinib 200ng/mL, imatinib 800ng/mL, sunitinib 80ng/mL, N-ethyl sunitinib 80ng/mL, erlotinib 1600ng/mL, tamoxifen 800ng/mL, capecitabine 400 ng/mL.
8. Use of the kit of any one of claims 1 to 7 for detecting the concentration of 11 anti-tumor drugs in serum by ultra performance liquid chromatography tandem mass spectrometry.
9. Use according to claim 8, characterized in that: the internal standard solution and the protein precipitant are firstly mixed to prepare the protein precipitant containing the internal standard, and then the protein precipitant is used for ultra-high performance liquid chromatography detection.
10. Use according to claim 9, characterized in that: the volume ratio of the internal standard solution to the protein precipitator is 0.1-0.3: 19.9-19.7.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011622833.3A CN112684057A (en) | 2020-12-31 | 2020-12-31 | Kit for detecting concentration of 11 anti-tumor drugs in serum and application thereof |
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| CN202011622833.3A CN112684057A (en) | 2020-12-31 | 2020-12-31 | Kit for detecting concentration of 11 anti-tumor drugs in serum and application thereof |
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Cited By (3)
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| CN114994213A (en) * | 2022-06-28 | 2022-09-02 | 北京赛诺浦生物技术有限公司 | Kit and method for determining blood concentration of anti-tumor drug tyrosine kinase inhibition in human plasma |
| CN115112785A (en) * | 2022-05-20 | 2022-09-27 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Human urine anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit |
| WO2023221472A1 (en) * | 2022-05-20 | 2023-11-23 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Tandem mass spectrometry detection kit for human plasma anti-hepatoma tyrosine kinase inhibitor |
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| CN115112785A (en) * | 2022-05-20 | 2022-09-27 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Human urine anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit |
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| CN114994213A (en) * | 2022-06-28 | 2022-09-02 | 北京赛诺浦生物技术有限公司 | Kit and method for determining blood concentration of anti-tumor drug tyrosine kinase inhibition in human plasma |
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Application publication date: 20210420 |