A method for detecting the concentration of 11 anti-tumor drugs in serum

Abstract

The present invention discloses a method for detecting the concentration of 11 anti-tumor drugs in serum, and belongs to the technical field of blood detection. The method uses ultra-high performance liquid chromatography tandem mass spectrometry to detect the concentration of 11 anti-tumor drugs in pretreated serum. The analyte is first separated from the serum matrix by ultra-high performance liquid chromatography, and then the mass spectrometry internal standard quantitative method is used to calculate the content of the 11 anti-tumor drugs. The method of the present invention has high sensitivity, strong specificity, accuracy and simple pretreatment process. The separation and detection of 11 anti-tumor drugs in serum can be completed within 6.5 minutes. The accuracy and precision basically meet the requirements, and can be used for the quantitative analysis of 11 anti-tumor drugs in serum in clinical practice, providing a simple and rapid detection method for clinical concentration monitoring of 11 anti-tumor drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of blood detection, and in particular relates to a method for detecting the concentrations of 11 anti-tumor drugs in serum. Background Art

[0002] At present, malignant tumors are one of the major diseases that threaten human life and health. The disease has the characteristics of high morbidity and high mortality. Due to the high incidence of cancer, the combined use of drugs is increasing. Therefore, the blood concentration monitoring of cancer drugs is of great significance.

[0003] Capecitabine is suitable for advanced primary or metastatic breast cancer. After oral administration, it is rapidly absorbed through the intestinal mucosa and then converted into an inactive intermediate 5'-deoxy-5'fluorocytidine by carboxylesterase in the liver. It is then converted into 5'-deoxy-5'fluorouridine by cytidine deaminase in the liver and tumor tissue, and finally catalyzed into fluorouracil (5-FU) in the tumor tissue by thymidine phosphorylase. Afatinib is a new generation of oral small molecule tyrosine kinase inhibitor (TKI) and the first irreversible ErbB family blocker. It can act on the entire ErbB family including EGFR. Unlike the first generation of reversible EGFR TKI, afatinib will irreversibly bind to EGFR, thereby achieving the purpose of shutting down cancer cell signaling pathways and inhibiting tumor growth. Tamoxifen is a synthetic anti-estrogen drug. It is similar in structure to estrogen and can compete with estradiol for estrogen receptors, form a stable complex with estrogen receptors, and be transported into the nucleus to prevent the opening of chromosomal genes, thereby inhibiting the growth and development of cancer cells. Gefitinib is an oral epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitor (a small molecule compound). The inhibition of EGFR-TK can hinder tumor growth, metastasis and angiogenesis, and increase tumor cell apoptosis. Imatinib, a tyrosine kinase inhibitor, is a small molecule protein kinase inhibitor that has the function of blocking one or more protein kinases. It is clinically used to treat chronic myeloid leukemia and malignant gastrointestinal stromal tumors. Sunitinib is a new multi-targeted oral drug for the treatment of tumors. It is used to treat gastrointestinal stromal tumors and metastatic renal cell carcinoma that do not respond to or cannot tolerate standard therapy. Erlotinib is a targeted therapy drug that can specifically target tumor cells, inhibit tumor formation and growth, and inhibit tumor growth by inhibiting the activity of tyrosine kinase. Tyrosine kinase is one of the important components in EGFR cells. It is used as a third-line treatment for locally advanced or metastatic non-small cell lung cancer that has failed two or more chemotherapy regimens. Icotinib hydrochloride is the first small molecule targeted anticancer drug in my country. It is a highly effective and specific epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) and is suitable for the second-line treatment of advanced non-small cell lung cancer. Apatinib is suitable for the third-line and above treatment of patients with advanced gastric adenocarcinoma or gastroesophageal junction adenocarcinoma. Crizotinib is a tyrosine kinase receptor inhibitor, including ALK, hepatocyte growth factor receptor (HGFR, c-Met), ROS1 (c-cos) and RON. Translocation can promote the expression of oncogenic fusion proteins caused by the ALK gene. The formation of ALK fusion proteins can cause activation and dysregulation of gene expression and signals, thereby promoting the proliferation and survival of tumor cells expressing these proteins.

[0004] At present, the main method for detecting the concentration of anti-tumor drugs is high performance liquid chromatography-tandem mass spectrometry. For example, patent CN108828077A discloses a kit for simultaneously detecting capecitabine and its metabolites in plasma, as well as its detection method and application. This method has certain defects: first, only one drug and its metabolites are detected. Since most patients will take drugs in combination with other drugs, its clinical significance is limited; second, the quantitative lower limit of capecitabine in this method is high, which has certain limitations on drug guidance. For example, an article entitled "An LC-MS / MS method for rapid and sensitive high-throughput simultaneous determination of various protein kinase inhibitors in human plasma" detected 5 kinds of protein kinase inhibitor drugs in human plasma based on LC-MS / MS. The sample volume of this method is 300μL, and the pretreatment adopts a more complex liquid-liquid extraction method. Even so, the quantitative lower limit of afatinib is 5ng / mL, and the trough concentration of afatinib after clinical administration is lower than 5ng / mL, so its clinical application is very limited. Summary of the invention

[0005] In view of the above-mentioned deficiencies of the prior art, the present invention provides a simple, efficient and reliable method for detecting the concentrations of 11 anti-tumor drugs in serum, which can better meet the needs of clinical application compared with the existing detection methods.

[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0007] A method for detecting the concentration of 11 anti-tumor drugs in serum, wherein the serum sample to be tested is pretreated, and then the test substance is separated from the serum matrix by ultra-high performance liquid chromatography, and then the mass spectrometry internal standard quantitative method is used to calculate the content of the 11 anti-tumor drugs according to the established calibration curve;

[0008] The pretreatment is to add a protein precipitant containing an internal standard to the serum sample to be tested, and take the supernatant after oscillation and centrifugation. The protein precipitant is a mixed solution of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 1:1-3;

[0009] The calibration curve is established with the concentration ratio of the standard to the internal standard as the X-axis and the peak area ratio of the standard to the internal standard as the Y-axis;

[0010] The 11 anti-tumor drugs are: capecitabine (CPT), afatinib (AFT), tamoxifen (TMX), gefitinib (GFT), imatinib (IMT), sunitinib (SNT), erlotinib (ELT), N-desethyl sunitinib (NSNT), icotinib hydrochloride (ICT), apatinib (APT) and crizotinib (CZP);

[0011] The chromatographic conditions used were as follows:

[0012] (1) Ultra-high performance liquid chromatography conditions:

[0013] Mobile phase A is a mixed solution of 0.01% to 0.2% formic acid and 1mM to 3mM ammonium acetate aqueous solution, and mobile phase B is acetonitrile;

[0014] The chromatographic column was Phenomenex Kinetex XB-C18, model 3.0×50 mm, 2.6 μm;

[0015] The method of gradient elution is adopted in which mobile phase A and mobile phase B are mixed as the mobile phase, the initial ratio of mobile phase A and mobile phase B is 85-100:25-0, and the gradient elution process is as follows: within 0-1.0 minute, the volume ratio of mobile phase A and mobile phase B is gradually changed from 85-100:25-0 to 70:30 at a uniform speed, within 1.0-3.0 minute, the volume ratio of mobile phase A and mobile phase B is gradually changed from 70:30 to 2:98 at a uniform speed, within 3.0-4.0 minute, the volume ratio of mobile phase A and mobile phase B is maintained at 2:98, within 4.0-6.5 minute, the volume ratio of mobile phase A and mobile phase B is changed from 2:98 to the initial ratio, and the collection time of each sample is 6.5 minutes;

[0016] The flow rate was 0.2 to 0.4 mL / min, the column temperature was 40 to 50 °C, and the injection volume was 0.2 to 2 μL;

[0017] (2) Mass spectrometry conditions:

[0018] In the electrospray ionization positive ion detection mode, the mass spectrometry scanning mode with multiple reaction monitoring was adopted, the capillary voltage was 3.0 kV (ESI+); the drying gas temperature was 300°C; the nebulizing gas pressure was 45 psi, the sheath gas temperature was 350°C, and the sheath gas flow rate was 11 L / min.

[0019] Furthermore, the internal standards corresponding to the 11 anti-tumor drugs are: capecitabine-d11 (CPT-d11), afatinib-d6 (AFT-d6), tamoxifen-d5 (TMX-d5), gefitinib-d3 (GFT-d3), imatinib-d8 (IMT-d8), sunitinib-d4 (SNT-d4), N-desethylsunitinib-d5 (NSNT-d5), apatinib-d8 (APT-d8) and crizotinib-d5 (CZP-d5).

[0020] Furthermore, the mobile phase A is a mixed solution of 0.1% formic acid and 2 mM ammonium acetate aqueous solution, the mobile phase B is acetonitrile, and the initial ratio of the mobile phase A to the mobile phase B is 99:1.

[0021] Furthermore, the flow rate is 0.3 mL / min, the column temperature is 45° C., and the injection volume is 1 μL.

[0022] Furthermore, the volume ratio of methanol to acetonitrile in the protein precipitant is 1:2.

[0023] Furthermore, the specific process of the pretreatment is: take 50 μL of serum into a 1.5 mL centrifuge tube, add 200 μL of protein precipitant containing internal standard, oscillate for 3 to 10 minutes, centrifuge at 12000 to 15000 r / min, 4 to 20° C. for 4 to 10 minutes, and take the supernatant.

[0024] Furthermore, the protein precipitant containing the internal standard is obtained by adding 20 μL of the internal standard solution to 19.98 mL of the protein precipitant, and the concentrations of the internal standards in the internal standard solution are: capecitabine-d11 1000 ng / mL, afatinib-d6 5000 ng / mL, tamoxifen-d5 500 ng / mL, gefitinib-d3 2000 ng / mL, imatinib-d8 5000 ng / mL, sunitinib-d4 400 ng / mL, N-desethylsunitinib-d5 400 ng / mL, apatinib-d8 2000 ng / mL and crizotinib-d5 2000 ng / mL.

[0025] The technical solution of the present invention has the following advantages:

[0026] The present invention provides a method for detecting the concentrations of 11 anti-tumor drugs in serum, which has high sensitivity, strong specificity, accuracy and simple pretreatment process. The separation and detection of 11 anti-tumor drugs in serum can be completed within 6.5 minutes, and the accuracy and precision basically meet the requirements. The method can be used for the quantitative analysis of 11 anti-tumor drugs in serum clinically, and provides a simple and rapid detection method for clinical concentration monitoring of 11 anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The extracted ion current spectra of 11 anti-tumor drug standards.

[0028] Figure 2 The extracted ion current spectra of 11 anti-tumor drugs in serum. DETAILED DESCRIPTION

[0029] The present invention provides a method for detecting the concentrations of 11 anti-tumor drugs in serum. After the serum sample is pretreated, it is shaken and centrifuged, and the supernatant is sampled. The concentrations of the 11 anti-tumor drugs in the pretreated serum are detected by ultra-high performance liquid chromatography tandem mass spectrometry. The ultra-high performance liquid chromatography is used to separate the analyte from the serum matrix, and then a mass spectrometry internal standard quantitative method is used, with the concentration ratio of the standard to the internal standard as the X-axis and the peak area ratio of the standard to the internal standard as the Y-axis, to establish a calibration curve, and calculate the contents of the 11 anti-tumor drugs.

[0030] The 11 anti-tumor drugs are: capecitabine (CPT), afatinib (AFT), tamoxifen (TMX), gefitinib (GFT), imatinib (IMT), sunitinib (SNT), erlotinib (ELT), N-desethyl sunitinib (NSNT), icotinib hydrochloride (ICT), apatinib (APT) and crizotinib (CZP);

[0031] The internal standards corresponding to the 11 anti-tumor drugs are: capecitabine-d11 (CPT-d11), afatinib-d6 (AFT-d6), tamoxifen-d5 (TMX-d5), gefitinib-d3 (GFT-d3), imatinib-d8 (IMT-d8), sunitinib-d4 (SNT-d4), N-desethylsunitinib-d5 (NSNT-d5), apatinib-d8 (APT-d8) and crizotinib-d5 (CZP-d5).

[0032] The specific chromatographic conditions are:

[0033] (1) Ultra-high performance liquid chromatography conditions:

[0034] Mobile phase A: 0.01% to 0.2% formic acid, 1mM to 3mM ammonium acetate aqueous solution; Mobile phase B: acetonitrile;

[0035] Chromatographic column model: Phenomenex Kinetex XB-C18 (3.0×50mm, 2.6μm);

[0036] The method of gradient elution is carried out by using mobile phase A and mobile phase B as a mixed mobile phase, wherein the initial ratio of mobile phase A and mobile phase B is 85-100:25-0; the gradient elution process is as follows: within 0-1.0 minutes, the volume ratio of mobile phase A and mobile phase B is gradually changed from 85-100:25-0 to 70:30 at a uniform speed; within 1.0-3.0 minutes, the volume ratio of mobile phase A and mobile phase B is gradually changed from 70:30 to 2:98 at a uniform speed; within 3.0-4.0 minutes, the volume ratio of mobile phase A and mobile phase B is maintained at 2:98, and within 4.0-6.5 minutes, the volume ratio of mobile phase A and mobile phase B is changed from 2:98 to the initial ratio, and the collection time of each sample is 6.5 minutes;

[0037] (2) Mass spectrometry conditions:

[0038] In the electrospray ionization positive ion detection mode, the mass spectrometry scanning mode with multiple reaction monitoring was adopted, the capillary voltage was 3.0 kV (ESI+); the drying gas temperature was 300°C; the nebulizing gas pressure was 45 psi, the sheath gas temperature was 350°C, and the sheath gas flow rate was 11 L / min; each target and its corresponding internal standard were monitored simultaneously.

[0039] In order to improve the selectivity of chromatographic separation, it is possible to consider adjusting the polarity of the mobile phase. The present invention adds formic acid and ammonium acetate to the mobile phase A, which can effectively improve the ionization efficiency of the target compound. Under the cooperation of other conditions, the sensitivity of detecting 11 anti-tumor drugs in serum by the LC-MS / MS method in the prior art is higher, the pretreatment process is simple, the cost is low, and the sensitivity is high and the specificity is strong. The separation and detection of 11 anti-tumor drugs are completed within 6.5 minutes. In a preferred embodiment, the mobile phase A is 0.01% to 0.2% formic acid, 1mM to 3mM ammonium acetate aqueous solution. Without affecting the effect of the present invention, preferably, the mobile phase A is 0.1% formic acid, 2mM ammonium acetate aqueous solution.

[0040] In chromatography, the selection of chromatographic columns is very important, and the requirements for chromatographic columns are: high column efficiency, good selectivity, fast analysis speed, etc. The present invention uses acetonitrile and 0.01% to 0.2% formic acid, 1mM to 3mM ammonium acetate aqueous solution as mobile phases, and the chromatographic column model is Phenomenex Kinetex XB-C18 (3.0×50mm, 2.6μm). Under the cooperation of other conditions, endogenous substances do not interfere with the determination of samples, and the sensitivity is high, the specificity is strong, and the accuracy and precision basically meet the requirements.

[0041] When using the internal standard method, the selection of the internal standard is a very important task. The ideal internal standard should be able to be added to the sample in an accurate and known amount, and have the same or as consistent physicochemical properties, chromatographic behavior and response characteristics as the sample being analyzed; under the conditions of chromatographic analysis, the internal standard must be able to be fully separated from the components in the sample. The present invention uses an isotope internal standard, and the internal standard and the analyte have the same chemical properties and matrix effect, and the reproducibility and accuracy of measuring the concentrations of 11 anti-tumor drugs in serum are good.

[0042] In a preferred embodiment, the initial ratio of mobile phase A to mobile phase B is 85-100:25-0. More preferably, the initial ratio of mobile phase A to mobile phase B is 99:1.

[0043] In a preferred embodiment, the flow rate is 0.2-0.4 mL / min, preferably 0.3 mL / min.

[0044] Furthermore, the column temperature is 40-50°C, preferably 45°C.

[0045] In one embodiment, the injection volume is 0.2 to 2 μL, preferably 1 μL.

[0046] In a preferred embodiment, when ultra-high performance liquid chromatography tandem mass spectrometry is used to detect 11 anti-tumor drugs in pre-treated serum, the specific chromatographic conditions are:

[0047] (1) Ultra-high performance liquid chromatography conditions:

[0048] Mobile phase A: 0.1% formic acid, 2 mM ammonium acetate aqueous solution; Mobile phase B: acetonitrile;

[0049] Chromatographic column model: Phenomenex Kinetex XB-C18 (3.0×50mm, 2.6μm);

[0050] The mobile phase A and the mobile phase B are used as a mixed mobile phase for gradient elution, and the initial ratio of the mobile phase A and the mobile phase B is 99:1; the gradient elution process is as follows: within 0-1.0 minutes, the volume ratio of the mobile phase A and the mobile phase B is gradually changed from 99:1 to 70:30 at a uniform speed; within 1.0-3.0 minutes, the volume ratio of the mobile phase A and the mobile phase B is gradually changed from 70:30 to 2:98 at a uniform speed; within 3.0-4.0 minutes, the volume ratio of the mobile phase A and the mobile phase B is maintained at 2:98, and within 4.0-6.5 minutes, the volume ratio of the mobile phase A and the mobile phase B is changed from 2:98 to the initial ratio, and the collection time of each sample is 6.5 minutes; the gradient elution method is specifically shown in Table 1; the flow rate is 0.3 mL / min, the column temperature is 45°C, and the injection volume is 1 μL.

[0051] Table 1 Mobile phase gradient elution parameters

[0052] 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

[0053] (2) Mass spectrometry conditions:

[0054] In the electrospray ionization positive ion detection mode, the mass spectrometry scanning mode of multiple reaction monitoring was adopted, the capillary voltage was 3.0 kV (ESI+); the drying gas temperature was 300°C; the nebulizing gas pressure was 45 psi, the sheath gas temperature was 350°C, and the sheath gas flow rate was 11 L / min; each target and its corresponding internal standard were monitored simultaneously; the mass spectrometry source parameters are shown in Table 2, and each target and its corresponding internal standard were monitored simultaneously. The mass spectrometry parameters of each target are shown in Table 3.

[0055] Table 2 Mass spectrometry source parameters

[0056] project parameter Capillary voltage(kV) 3.0 Drying gas temperature (℃) 300 Atomizer pressure (psi) 45 Sheath gas temperature (℃) 350 Sheath gas flow rate (L / min) 11

[0057] Table 3 Mass spectrometry parameters for detection of 11 anti-tumor drugs

[0058] Compound Precursor ion Product ions Declustering voltage (V) Impact voltage(V) ESI(+ / -) CPT 360.32 129.86 38 30 + CPT-d11 371.32 130.86 38 30 + NSNT 371.10 282.98 6 22 + NSNT-d5 376.23 282.97 10 22 + TMX 372.23 71.91 2 8 + TMX-d5 377.23 71.91 36 22 + ICT 392.23 303.98 6 16 + ELT 394.23 277.96 26 15 + APT 398.23 211.99 6 10 + APT-d8 406.30 211.99 10 24 + SNT 399.23 282.97 6 24 + SNT-d4 403.23 282.99 24 22 + GFT 447.23 127.97 76 6 + GFT-d8 450.17 127.97 72 18 + CZ 450.20 259.77 40 20 + CZP-d5 455.27 265.03 78 22 + AFT 486.30 371.00 10 24 + AFT-d6 492.36 371.01 2 24 + IMT 494.40 394.11 2 15 + IMT-d8 502.46 394.12 2 24 +

[0059] The serum mentioned in the present invention is human or animal serum.

[0060] The pretreated serum mentioned in the present invention is prepared according to the following method: add a protein precipitant containing an internal standard to the serum, and then oscillate and centrifuge to take the supernatant and put it on the machine. The protein precipitant containing the internal standard is prepared by mixing an internal standard solution and a protein precipitant, and the volume ratio of the internal standard solution to the protein precipitant is 0.1-0.3:19.9-19.7. The protein precipitant 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.

[0061] In a preferred embodiment, the pretreated serum mentioned in the present invention is prepared according to the following method: take 50 μL of serum in a 1.5 mL centrifuge tube, add 200 μL of protein precipitant containing internal standard, oscillate for 3 to 10 minutes, centrifuge at 12000 to 15000 r / min, 4 to 20° C. for 4 to 10 minutes, take the supernatant into an injection bottle for injection; the volume ratio of methanol to acetonitrile in the protein precipitant is 1:2.

[0062] In a more preferred embodiment, the pretreated serum mentioned in the present invention is prepared as follows: 50 μL of serum is placed in a 1.5 mL centrifuge tube, 200 μL of a protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:2) is added thereto, shaken for 5 minutes, centrifuged at 14000 r / min, 15° C. for 5 minutes, 60 μL of the supernatant is taken into an injection bottle, and 1 μL is injected.

[0063] In one embodiment, the protein precipitant containing an internal standard mentioned in the present invention is prepared according to the following method:

[0064] The following internal standard stock solutions were prepared with methanol and water: afatinib-d6 1 mg / mL, apatinib-d8 1 mg / mL, gefitinib-d3 1 mg / mL, imatinib-d8 1 mg / mL, sunitinib-d4 0.1 mg / mL, N-desethylsunitinib-d5 0.1 mg / mL, tamoxifen-d5 0.1 mg / mL, capecitabine-d11 0.1 mg / mL, and crizotinib-d5 1 mg / mL;

[0065] Take out the internal standard stock solutions: afatinib-d6 2.5μL, apatinib-d8 1μL, gefitinib-d3 1μL, imatinib-d82.5μL, sunitinib-d4 2μL, N-desethylsunitinib-d5 2μL, tamoxifen-d5 2.5μL, capecitabine-d11 5μL and crizotinib-d5 1μL; add to 480.5μL methanol water to obtain 500μL standard stock solution;

[0066] Take 20 μL of the internal standard solution and add it to 19.98 mL of protein precipitant to obtain a protein precipitant containing the internal standard.

[0067] In one embodiment, the protein precipitant is a mixed solution of methanol and acetonitrile; preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1:1 to 3. More preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1:2.

[0068] In a preferred embodiment, the protein starch containing internal standard mentioned in the present invention is prepared according to the following method:

[0069] The following internal standard stock solutions were prepared with methanol water: afatinib-d6 1 mg / mL, apatinib-d8 1 mg / mL, gefitinib-d3 1 mg / mL, imatinib-d8 1 mg / mL, sunitinib-d4 0.1 mg / mL, N-desethylsunitinib-d5 0.1 mg / mL, tamoxifen-d5 0.1 mg / mL, capecitabine-d11 0.1 mg / mL, and crizotinib-d5 1 mg / mL;

[0070] Pipette the internal standard stock solutions: afatinib-d6 2.5 μL, apatinib-d8 1 μL, gefitinib-d3 1 μL, imatinib-d8 2.5 μL, sunitinib-d4 2 μL, N-desethylsunitinib-d5 2 μL, tamoxifen-d5 2.5 μL, capecitabine-d11 5 μL, and crizotinib-d5 1 μL; add to 480.5 μL of methanol water to obtain 500 μL of standard stock solution.

[0071] Take 20 μL of the above internal standard solution and add it to 19.98 mL of protein precipitant to obtain a protein precipitant containing internal standard (methanol and acetonitrile are 1:2). The protein precipitant contains: afatinib-d6 5 ng / mL, apatinib-d8 2 ng / mL, gefitinib-d3 2 ng / mL, imatinib-d8 5 ng / mL, sunitinib-d4 0.4 ng / mL, N-desethyl sunitinib-d5 0.4 ng / mL, tamoxifen-d5 0.5 ng / mL, capecitabine-d11 1 ng / mL and crizotinib-d5 2 ng / mL.

[0072] In a more preferred embodiment, the protein precipitant containing an internal standard mentioned in the present invention is prepared according to the following method:

[0073] Prepare internal standard mother solution with 50% methanol water, transfer the standard mother solution to 480.5μL 50% methanol water, mix well to get 500μL internal standard solution, take 20μL of the mixed internal standard solution and add it to 19.98mL methanol and acetonitrile mixed solution (the volume ratio of methanol to acetonitrile is 1:2), and get the protein precipitant containing internal standard, the concentration is shown in Table 4 below. It is recommended to store it in aliquots and freeze it in a -80℃ refrigerator for immediate use.

[0074] Table 4 Preparation of protein precipitant containing internal standard

[0075]

[0076] In one embodiment, the standard substance mentioned in the present invention is prepared as follows:

[0077] The following concentrations of standard stock solutions were prepared: afatinib 0.1 mg / mL, apatinib 0.1 mg / mL, gefitinib 5 mg / mL, icotinib hydrochloride 2 mg / mL, crizotinib 0.1 mg / mL, imatinib 2 mg / mL, sunitinib 0.1 mg / mL, N-desethylsunitinib 0.1 mg / mL, erlotinib 2 mg / mL, tamoxifen 2 mg / mL, and capecitabine 2 mg / mL;

[0078] Pipette the following standard stock solutions: 20 μL of afatinib, 40 μL of apatinib, 4 μL of gefitinib, 5 μL of icotinib hydrochloride, 50 μL of crizotinib, 10 μL of imatinib, 20 μL of sunitinib, 20 μL of N-desethylsunitinib, 20 μL of erlotinib, 10 μL of tamoxifen and 5 μL of capecitabine; add to 296 μL of methanol water to obtain 500 μL of standard stock solution.

[0079] The mixed standard stock solution was prepared into seven calibration solutions with different concentrations using blank serum matrix. The seven concentrations of the calibration solution were:

[0080] The seven concentration points of afatinib, sunitinib, and N-desethyl sunitinib are: 0.4 ng / mL, 1 ng / mL, 2 ng / mL, 10 ng / mL, 20 ng / mL, 100 ng / mL, and 200 ng / mL;

[0081] The seven concentration points of apatinib are: 0.8ng / mL, 2ng / mL, 4ng / mL, 20ng / mL, 40ng / mL, 200ng / mL, 400ng / mL;

[0082] The seven concentration points of crizotinib are: 1ng / mL, 2.5ng / mL, 5ng / mL, 25ng / mL, 50ng / mL, 250ng / mL, and 500ng / mL;

[0083] The seven concentration points of icotinib hydrochloride and capecitabine are: 2ng / mL, 5ng / mL, 10ng / mL, 50ng / mL, 100ng / mL, 500ng / mL, 1000ng / mL;

[0084] The seven concentration points of gefitinib, imatinib, and tamoxifen are: 4 ng / mL, 10 ng / mL, 20 ng / mL, 100 ng / mL, 200 ng / mL, 1000 ng / mL, and 2000 ng / mL;

[0085] The seven concentration points of erlotinib are: 8ng / mL, 20ng / mL, 40ng / mL, 200ng / mL, 400ng / mL, 2000ng / mL, and 4000ng / mL.

[0086] In a preferred embodiment, the blank serum matrix is ​​blank serum without the target drug.

[0087] In a preferred embodiment, the standard solution is prepared as follows:

[0088] The standard stock solutions of 11 anti-tumor drugs were pipetted and added to 296 μL of 50% methanol water to obtain 500 μL of standard stock solutions. The concentrations are shown in Table 5 below.

[0089] Table 5 Standard stock solutions

[0090]

[0091] The present invention prepares the standard stock solution into seven calibration solutions at different concentrations using a blank serum matrix (blank serum without the target drug), and the preparation process is as follows:

[0092] Take 10 μL of the standard stock solution and add it to 190 μL of blank serum matrix as the first high concentration point; take 50 μL of the first high concentration point and dilute it with 50 μL of blank serum matrix to obtain the second high concentration point; take the first high concentration point and dilute it with 9 times the volume of blank serum matrix to obtain the third high concentration point; take the second high concentration point and dilute it with 9 times the volume of blank serum matrix to obtain the fourth high concentration point; take the third high concentration point and dilute it with 9 times the volume of blank serum matrix to obtain the fifth high concentration point; take the fourth high concentration point and dilute it with 9 times the volume of blank serum matrix to obtain the sixth high concentration point; take the fifth high concentration point and dilute it with 5 times the volume of blank serum matrix to obtain the seventh high concentration point.

[0093] The present invention adopts the gradient dilution method to prepare the standard curve. After taking out the standard solution from the -80°C refrigerator, vortex for 10 seconds, use the standard solution to prepare the highest concentration point of the standard curve within 2 minutes, and store it at -80°C after preparation. The specific process is shown in Table 6 below.

[0094] Table 6 Preparation of standard curve

[0095] Marking song Pipette solution (μL) Blank serum matrix (μL) S7 Mixed standard stock solution 10 190 S6 S7 50 50 S5 S7 20 180 S4 S6 20 180 S3 S5 20 180 S2 S4 20 180 S1 S3 25 125

[0096] Take 50 μL of each concentration point of the seven different calibration samples in a 1.5 mL centrifuge tube, add 200 μL of protein precipitant containing internal standard (the volume ratio of methanol to acetonitrile is 1:2), oscillate for 5 minutes, centrifuge at 14000 r / min, 15°C for 5 minutes, take 60 μL of the supernatant into the injection bottle, and inject 1 uL.

[0097] The present invention also includes preparing a quality control product, wherein the quality control product is a blank serum containing 11 anti-tumor drugs, and is divided into three concentrations of low, medium and high, namely QC(L), QC(M) and QC(H);

[0098] QC (L) is the above standard stock solution diluted to 5000 times with blank serum matrix.

[0099] QC (M) is the above standard stock solution diluted 500 times with blank serum matrix.

[0100] QC(H) is the above standard stock solution diluted 50 times with blank serum matrix.

[0101] Preferably, the blank serum matrix is ​​blank serum without the target drug.

[0102] In a preferred embodiment, the quality control product is prepared as follows: the above standard stock solution is prepared into three different concentrations of QC (L), QC (M), and QC (H) with blank serum without the target drug, as shown in Table 7.

[0103] Table 7 Corresponding concentrations of quality control products (unit: ng / mL)

[0104] Compound ID Compound QC(L) QC(M) QC(H) 1 AFT 0.8 8 80 2 APT 1.6 16 160 3 GFT 8 80 800 4 ICT 4 40 400 5 CZ 2 20 200 6 IMT 8 80 800 7 SNT 0.8 8 80 8 NSNT 0.8 8 80 9 ELT 16 160 1600 10 TMX 8 80 800 11 CPT 4 40 400

[0105] QC(L) includes: afatinib 0.8ng / mL, apatinib 1.6ng / mL, gefitinib 8ng / mL, icotinib hydrochloride 4ng / mL, crizotinib 2ng / mL, imatinib 8ng / mL, sunitinib 0.8ng / mL, N-desethylsunitinib 0.8ng / mL, erlotinib 16ng / mL, tamoxifen 8ng / mL, and capecitabine 4ng / mL.

[0106] QC(M) includes: afatinib 8ng / mL, apatinib 16ng / mL, gefitinib 80ng / mL, icotinib hydrochloride 40ng / mL, crizotinib 20ng / mL, imatinib 80ng / mL, sunitinib 8ng / mL, N-desethylsunitinib 8ng / mL, erlotinib 160ng / mL, tamoxifen 80ng / mL, and capecitabine 40ng / mL.

[0107] QC(H) includes: afatinib 80ng / mL, apatinib 160ng / mL, gefitinib 800ng / mL, icotinib hydrochloride 400ng / mL, crizotinib 200ng / mL, imatinib 800ng / mL, sunitinib 80ng / mL, N-desethylsunitinib 80ng / mL, erlotinib 1600ng / mL, tamoxifen 800ng / mL, capecitabine 400ng / mL.

[0108] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, but it should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, the modification or replacement of the method, steps or conditions of the present invention all belong to the scope of the present invention. The experimental methods and reagents without specifying the specific conditions in the examples are all in accordance with the conventional conditions in the art.

[0109] Example 1

[0110] 1. Experimental Materials and Instruments

[0111] 1. Materials

[0112] The samples came from serum samples collected from the outpatient department of Nanjing Drum Tower Hospital in October 2019.

[0113] (1) Instruments: Qlife Lab 9000plus triple quadrupole mass spectrometer (PinSheng Medical); Qlife Lab 9000 ultra-high performance liquid chromatography system (with G7167A autosampler, PinSheng Medical); SCILOGEX D2012 high-speed desktop centrifuge (USA); ultrapure water meter (ELGA LabWater, UK); multi-tube vortex mixer (Vortex genie2, USA); adjustable pipettes (Eppendorf 0.5-10 μL, 10-100 μL, 100-1000 μL); glass instruments, measuring cylinders, etc.

[0114] (2) Reagents and 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) Chromatographic column model: Phenomenex Kinetex XB-C18 (3.0×50 mm, 2.6 μm).

[0115] (3) Standards: Standards and their corresponding internal standards are listed in Table 8 below.

[0116] Table 8 Standards and internal standards

[0117]

[0118]

[0119] (4) Quality control products: blank serum containing 11 anti-tumor drugs, with three concentrations of low, medium and high, namely QC(L), QC(M) and QC(H), as shown in Table 7.

[0120] 2. Liquid quality conditions

[0121] (1) Chromatographic conditions: Mobile phase A: 0.1% formic acid, 2 mM ammonium acetate aqueous solution; Mobile phase B: acetonitrile. Chromatographic column model: Phenomenex Kinetex XB-C18 (3.0×50 mm, 2.6 μm), gradient elution, see Table 1 for details. Flow rate: 0.3 mL / min, column temperature: 45°C, injection volume: 1 μL.

[0122] (2) In the electrospray ionization positive ion detection mode, the mass spectrometry scanning mode of multiple reaction monitoring was adopted, the capillary voltage was 3.0 kV (ESI+); the drying gas temperature was 300°C; the nebulizing gas pressure was 45 psi, the sheath gas temperature was 350°C, and the sheath gas flow rate was 11 L / min; each target and its corresponding internal standard were monitored simultaneously; the mass spectrometry source parameters are shown in Table 2, and each target and its corresponding internal standard were monitored simultaneously. The mass spectrometry parameters of each target are shown in Table 3.

[0123] 3. Experimental process

[0124] (1) Preparation of standard products:

[0125] Eleven anti-tumor drugs were prepared into standard stock solutions with the following concentrations: afatinib 0.1 mg / mL, apatinib 0.1 mg / mL, gefitinib 5 mg / mL, icotinib hydrochloride 2 mg / mL, crizotinib 0.1 mg / mL, imatinib 2 mg / mL, sunitinib 0.1 mg / mL, N-desethylsunitinib 0.1 mg / mL, erlotinib 2 mg / mL, tamoxifen 2 mg / mL, and capecitabine 2 mg / mL;

[0126] Take the stock solutions of the standards: 20 μL afatinib, 40 μL apatinib, 4 μL gefitinib, 5 μL icotinib hydrochloride, 50 μL crizotinib, 10 μL imatinib, 20 μL sunitinib, 20 μL N-desethyl sunitinib, 20 μL erlotinib, 10 μL tamoxifen and 5 μL capecitabine; add to 296 μL 50% methanol water to obtain 500 μL standard stock solution. See Table 5 for details.

[0127] The above standard stock solution was prepared into calibrator solutions at seven different concentrations using blank serum matrix (blank serum without target drug), as shown in Table 6. The seven concentrations of the calibrator solutions are:

[0128] The seven concentration points of afatinib, sunitinib, and N-desethyl sunitinib are: 0.4 ng / mL, 1 ng / mL, 2 ng / mL, 10 ng / mL, 20 ng / mL, 100 ng / mL, and 200 ng / mL;

[0129] The seven concentration points of apatinib are: 0.8ng / mL, 2ng / mL, 4ng / mL, 20ng / mL, 40ng / mL, 200ng / mL, 400ng / mL;

[0130] The seven concentration points of crizotinib are: 1ng / mL, 2.5ng / mL, 5ng / mL, 25ng / mL, 50ng / mL, 250ng / mL, and 500ng / mL;

[0131] The seven concentration points of icotinib hydrochloride and capecitabine are: 2ng / mL, 5ng / mL, 10ng / mL, 50ng / mL, 100ng / mL, 500ng / mL, 1000ng / mL;

[0132] The seven concentration points of gefitinib, imatinib, and tamoxifen are: 4 ng / mL, 10 ng / mL, 20 ng / mL, 100 ng / mL, 200 ng / mL, 1000 ng / mL, and 2000 ng / mL;

[0133] The seven concentration points of erlotinib are: 8ng / mL, 20ng / mL, 40ng / mL, 200ng / mL, 400ng / mL, 2000ng / mL, and 4000ng / mL.

[0134] (2) Preparation of protein precipitant containing internal standard

[0135] The following internal standard stock solutions were prepared: afatinib-d6 1 mg / mL, apatinib-d8 1 mg / mL, gefitinib-d3 1 mg / mL, imatinib-d8 1 mg / mL, sunitinib-d4 0.1 mg / mL, N-desethylsunitinib-d5 0.1 mg / mL, tamoxifen-d5 0.1 mg / mL, capecitabine-d11 0.1 mg / mL, and crizotinib-d5 1 mg / mL;

[0136] Pipette the internal standard stock solutions: afatinib-d6 2.5μL, apatinib-d8 1μL, gefitinib-d3 1μL, imatinib-d82.5μL, sunitinib-d4 2μL, N-desethylsunitinib-d5 2μL, tamoxifen-d5 2.5μL, capecitabine-d11 5μL, and crizotinib-d5 1μL; add to 480.5μL. 500 μL of standard stock solution was obtained, including afatinib-d6 5000 ng / mL, apatinib-d8 2000 ng / mL, gefitinib-d3 2000 ng / mL, imatinib-d8 5000 ng / mL, sunitinib-d4 400 ng / mL, N-desethylsunitinib-d5 400 ng / mL, tamoxifen-d5 500 ng / mL, capecitabine-d11 1000 ng / mL and crizotinib-d5 2000 ng / mL.

[0137] Take 20 μL of the mixed internal standard solution and add it to 19.98 mL of protein precipitant (methanol and acetonitrile is 1:2) to obtain a protein precipitant containing the internal standard. The protein precipitant contains: afatinib-d6 5 ng / mL, apatinib-d8 2 ng / mL, gefitinib-d3 2 ng / mL, imatinib-d8 5 ng / mL, sunitinib-d4 0.4 ng / mL, N-desethyl sunitinib-d5 0.4 ng / mL, tamoxifen-d5 0.5 ng / mL, capecitabine-d11 1 ng / mL and crizotinib-d5 2 ng / mL.

[0138] (3) Preparation of quality control products:

[0139] The above standard stock solution was prepared into three different concentrations of QC (L), QC (M), and QC (H) with blank serum without the target drug, as shown in Table 7.

[0140] QC(L) includes: afatinib 0.8ng / mL, apatinib 1.6ng / mL, gefitinib 8ng / mL, icotinib hydrochloride 4ng / mL, crizotinib 2ng / mL, imatinib 8ng / mL, sunitinib 0.8ng / mL, N-desethylsunitinib 0.8ng / mL, erlotinib 16ng / mL, tamoxifen 8ng / mL, and capecitabine 4ng / mL.

[0141] QC(M) includes: afatinib 8ng / mL, apatinib 16ng / mL, gefitinib 80ng / mL, icotinib hydrochloride 40ng / mL, crizotinib 20ng / mL, imatinib 80ng / mL, sunitinib 8ng / mL, N-desethylsunitinib 8ng / mL, erlotinib 160ng / mL, tamoxifen 80ng / mL, and capecitabine 40ng / mL.

[0142] QC(H) includes: afatinib 80ng / mL, apatinib 160ng / mL, gefitinib 800ng / mL, icotinib hydrochloride 400ng / mL, crizotinib 200ng / mL, imatinib 800ng / mL, sunitinib 80ng / mL, N-desethylsunitinib 80ng / mL, erlotinib 1600ng / mL, tamoxifen 800ng / mL, capecitabine 400ng / mL.

[0143] (4) Sample processing

[0144] 1) Standard treatment: Take 50 μL of each concentration point of seven different calibration samples in a 1.5 mL centrifuge tube, add 200 μL of protein precipitant containing internal standard (the volume ratio of methanol to acetonitrile is 1:2), shake for 5 min, centrifuge at 14000 r / min, 15°C for 5 min, take 60 μL of supernatant into the injection bottle, and inject 1 μL.

[0145] 2) Serum sample pretreatment: Take 50 μL into a 1.5 mL centrifuge tube, add 200 μL of protein precipitant containing internal standard (the volume ratio of methanol to acetonitrile is 1:2), shake for 5 min, centrifuge at 14000 r / min, 15°C for 5 min, take 60 μL of supernatant into an injection bottle, and inject 1 μL.

[0146] 3) Quality control sample pretreatment: Take 50 μL of each quality control solution QC(L), QC(M), and QC(H) in a 1.5 mL centrifuge tube, and then perform the same pretreatment as the serum sample, which will not be repeated here.

[0147] IV. Method Validation

[0148] 1. Extracted ion current chromatogram: The peak shapes of the standard products and serum samples of 11 anti-tumor drugs are relatively symmetrical, and there is no interference from other peaks, indicating that good detection can be obtained under this condition. Figure 1 Extracted ion current spectra for 11 anti-tumor drug standards. Figure 2 Extracted ion current spectra of 11 anti-tumor drugs in serum.

[0149] 2. Calibration curve: The internal standard quantitative method was used, and the TargetLynx software was used to establish a calibration curve with the concentration ratio of the standard to the internal standard as the X-axis and the peak area ratio of the standard to the internal standard as the Y-axis, and the concentration of the analyte in serum was calculated. The linear fitting equations of the 11 anti-tumor drugs within their respective concentration ranges were linear, with correlation coefficients above 0.99, meeting the quantitative requirements, as shown in Table 9.

[0150] Table 9 Linear regression equations and linear correlation coefficients of 11 anti-tumor drugs

[0151]

[0152] 3. Accuracy investigation: The accuracy of the method was evaluated by the spike recovery test. A mixed blank serum sample was prepared, and three mixed standards of low, medium and high concentrations were added respectively. The same steps were repeated for five times. The results showed that the spike recovery rates of 11 anti-tumor drugs were between 96.16% and 107.13%, and the RSD of the five repeated tests was in the range of 1.45% and 6.68%. The statistical results are shown in Table 10.

[0153] Table 10 Recovery results of 11 anti-tumor drugs

[0154]

[0155] 4. Precision test: Take a blank serum sample without interference, add 11 anti-tumor drug standards of different concentrations to obtain serum samples of low, medium and high concentrations, repeat the process for 6 batches in one day, and continue for three days. The concentrations of 11 anti-tumor drugs were quantitatively determined by the internal standard method. The intra-batch precision was 2.34% to 13.90%. The samples were divided into 3 batches within three days, and the inter-batch precision was calculated to be 0.38% to 7.78%. The results are shown in Table 11.

[0156] Table 11 Intra-batch and inter-batch precision test results

[0157]

[0158]

[0159]

[0160]

[0161] The present invention uses ID-HPLC-MS / MS method to determine the concentration of 11 anti-tumor drugs in human serum. The peak time and ion pair of the target are detected at the same time, with high sensitivity. At the same time, the internal standard method can greatly eliminate matrix interference, and is not affected by the pretreatment process, sample volume and mobile phase conditions, and can achieve accurate quantification.

[0162] The accuracy of the method was evaluated by a spike recovery test. The results showed that the spike recoveries of 11 anti-tumor drugs were between 90.63% and 107.13%, and the RSDs of five repeated tests were between 1.45% and 8.55%, indicating good accuracy.

[0163] The reproducibility results of the method showed that the intra-batch precision of 11 anti-tumor drugs was 2.34% to 13.90%, and the inter-batch precision was 0.38% to 13.49%. The serum sample pretreatment process established was very simple, and the amount of serum used was only 50μL.

[0164] In summary, the detection method of the present invention has high sensitivity, strong specificity, accuracy and a relatively simple pretreatment process. The separation and detection of the compound can be completed within 6.5 minutes. The accuracy and precision meet the requirements. It can be used for the quantitative analysis of the concentrations of 11 anti-tumor drugs in serum in clinical practice, providing a reliable detection method for clinical treatment monitoring of the concentrations of 11 anti-tumor drugs.

[0165] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that it is still possible to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the concentrations of 11 anti-tumor drugs in serum, characterized in that: After pretreatment of the serum samples to be tested, ultra-high performance liquid chromatography was used to separate the analyte from the serum matrix, and then the mass spectrometry internal standard quantification method was used to calculate the contents of 11 anti-tumor drugs based on the established calibration curve; The 11 anti-tumor drugs are: capecitabine, afatinib, tamoxifen, gefitinib, imatinib, sunitinib, erlotinib, N-desethyl sunitinib, icotinib hydrochloride, apatinib and crizotinib; the internal standards corresponding to the 11 anti-tumor drugs are: capecitabine-d11, afatinib-d6, tamoxifen-d5, gefitinib-d3, imatinib-d8, sunitinib-d4, N-desethyl sunitinib-d5, apatinib-d8 and crizotinib-d5; The specific process of the pretreatment is: taking 50 μL of serum into a 1.5 mL centrifuge tube, adding 200 μL of a protein precipitant containing an internal standard thereto, shaking for 3 to 10 minutes, centrifuging at 12000 to 15000 r / min and 4 to 20° C. for 4 to 10 minutes, and taking the supernatant; the protein precipitant containing the internal standard is obtained by adding an internal standard solution to the protein precipitant, and the volume ratio of the internal standard solution to the protein precipitant is 0.02:19.98; the concentrations of the internal standards in the internal standard solution are: capecitabine-d11 1000 ng / mL, afatinib-d6 5000 ng / mL, tamoxifen-d5 500 ng / mL, gefitinib-d3 2000 ng / mL, imatinib-d8 5000 ng / mL, sunitinib-d4 400 ng / mL, N-desethyl sunitinib-d5 400ng / mL, apatinib-d8 2000ng / mL and crizotinib-d5 2000ng / mL; the protein precipitant is a mixed solution of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is 1:2; The calibration curve is established with the concentration ratio of the standard to the internal standard as the X-axis and the peak area ratio of the standard to the internal standard as the Y-axis; The chromatographic conditions used were as follows: (1) UPLC conditions: mobile phase A was a mixture of 0.1% formic acid and 2 mM ammonium acetate aqueous solution, mobile phase B was acetonitrile; chromatographic column was Phenomenex Kinetex XB-C18, model 3.0×50mm, 2.6μm; mobile phase A and mobile phase B were used as mixed mobile phases for gradient elution, the initial ratio of mobile phase A and mobile phase B was 99:1, and the gradient elution process was as follows: within 0-1.0 minutes, the volume ratio of mobile phase A and mobile phase B was gradually changed from 99:1 to 70:30 at a uniform rate, within 1.0-3.0 minutes, the volume ratio of mobile phase A and mobile phase B was gradually changed from 70:30 to 2:98 at a uniform rate, within 3.0-4.0 minutes, the volume ratio of mobile phase A and mobile phase B was maintained at 2:98, within 4.0-6.5 minutes, the volume ratio of mobile phase A and mobile phase B was changed from 2:98 to the initial ratio, and the collection time of each sample was 6.5 minutes; the flow rate was 0.3mL / min, the column temperature was 45℃, and the injection volume was 1μL; (2) Mass spectrometry conditions: In electrospray ionization positive ion detection mode, the mass spectrometry scanning mode with multiple reaction monitoring was adopted, the capillary voltage was 3.0 kV ESI+; the drying gas temperature was 300 °C; the nebulizing gas pressure was 45 psi, the sheath gas temperature was 350 °C, and the sheath gas flow rate was 11 L / min; The mass spectrometry parameters for the detection of the 11 anti-tumor drugs are as follows:

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