WO2023221487A1 - 一种人体尿液抗肝癌酪氨酸激酶抑制剂串联质谱检测试剂盒 - Google Patents

一种人体尿液抗肝癌酪氨酸激酶抑制剂串联质谱检测试剂盒 Download PDF

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WO2023221487A1
WO2023221487A1 PCT/CN2022/139272 CN2022139272W WO2023221487A1 WO 2023221487 A1 WO2023221487 A1 WO 2023221487A1 CN 2022139272 W CN2022139272 W CN 2022139272W WO 2023221487 A1 WO2023221487 A1 WO 2023221487A1
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solution
mass spectrometry
tivantinib
tandem mass
tyrosine kinase
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PCT/CN2022/139272
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French (fr)
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李挥
姜文
甄晓兰
哈婧
邢娣娣
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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)
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G01N30/724—Nebulising, aerosol formation or ionisation
    • G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray

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  • the invention belongs to the technical field of inhibitor detection, and is specifically a human urine anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit.
  • TKIs tyrosine kinase inhibitors
  • ATP adenosine triphosphate
  • TKIs have the characteristics of high selectivity and fewer adverse reactions. They are widely used in small cell lung cancer, non-small cell lung cancer, gastrointestinal stromal tumor, hepatocellular carcinoma, renal cancer and other cancers. At present, TKIs have made great progress in cancer treatment. The combined treatment of TKIs and hepatic artery chemoembolization is more effective than hepatic artery chemoembolization alone and can effectively reduce the incidence of adverse reactions.
  • Urine is a commonly used biological fluid in the study of small molecule metabolites.
  • the sample complexity is low, the pre-treatment is relatively simple, and metabolites are abundant in urine. Only a small amount of samples need to be prepared before analysis, and the collection is simple and non-invasive. Therefore, the present invention establishes a method for simultaneously detecting 9 TLIs in the urine of liver cancer patients based on human urine samples, which comprehensively covers the current TKIs with anti-liver cancer effects and is of great significance for clinical therapeutic drug monitoring.
  • LC-MS/MS combines the separation capabilities of chromatography and the analysis capabilities of mass spectrometry, and has the advantages of large dynamic range, fast analysis speed, high sensitivity, and low detection limit.
  • QuEChERS is an emerging pretreatment technology derived from dispersive solid phase extraction (dSPE). It has the advantages of high recovery rate and simple operation. It was initially used in the field of pesticide residues. In recent years, with the improvement and development of this technology, It is gradually applied to the analysis of metabolites and other compounds in biological matrices such as urine, plasma and tissue.
  • Hepatocellular carcinoma is a disease with poor prognosis, characterized by high morbidity and mortality and difficulty in early detection.
  • the nine tyrosine kinase inhibitors tested simultaneously by the present invention are all small molecule targeted drugs with anti-liver cancer effects.
  • molecular targeted therapy plays an irreplaceable role in the treatment of advanced liver cancer, the acquisition of drug resistance and drug toxicity are still inevitable, thus limiting the treatment of cancer. Therefore, it is necessary to monitor therapeutic drugs, assist in clinical medication guidance, adjust dosage in a timely manner, and improve drug efficacy and safety.
  • the present invention combines the QuEChERS pretreatment method with high-performance liquid chromatography tandem mass spectrometry to reduce the impact of matrix effects and compound instability on experimental results, thereby improving the quality of clinical detection.
  • the purpose of the present invention is to address the above problems and provide a human urine anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit, which is characterized in that it includes a calibrator, a quality control product, an internal standard solution, an extractant, and QuEChERS Purification materials, salting out agents, mobile phase A liquid, mobile phase B liquid, reconstitution solution, water-proof packaging bags and packaging boxes that separate and centrally package these reagent bottles or tubes.
  • calibrator use methanol diluent, while adding tivantinib, galunisrtib, sorafenib, lenvatinib, regorafenib, cabozantinib, anlotinib, apatinib and
  • the standard of gefitinib is prepared into calibrators A-G with 7 concentration dose points, in which the concentrations of tivantinib and galunisrtib are 2ng/mL, 5ng/mL, 1Ong/mL, 20ng/mL, and 50ng/mL respectively.
  • the concentrations of sorafenib, lenvatinib, regorafenib, cabozantinib, anlotinib, apatinib and gefitinib are 0.2ng/ mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 8ng/mL, 10ng/mL;
  • Quality control products Use methanol diluent, and add tivantinib, galunisrtib, sorafenib, lenvatinib, regorafenib, cabozantinib, anlotinib, apatinib and gifef at the same time
  • the standard substance of tivantinib was prepared into three concentrations of quality control products C1-C3, among which the concentrations of tivantinib and galunisrtib were 2ng/mL, 50ng/mL, and 100ng/mL respectively, and the concentrations of sorafenib and lenvatinib were respectively
  • concentrations of nib, regorafenib, cabozantinib, anlotinib, apatinib and gefitinib are 0.2ng/mL, 5ng/mL and 10ng/mL respectively;
  • Extractant 1-3mL acetonitrile
  • QuEChERS purification material 30-60mg C18, packaged in a water-proof packaging bag;
  • Salting out agent 10-40mg sodium chloride, 50-100mg anhydrous magnesium sulfate, sealed in a water-proof packaging bag;
  • Mobile phase B prepared from formic acid and purified water, the content of formic acid is 0.05%-0.2%;
  • the dosage of sodium chloride in the salting out agent is 20 mg, and the dosage of anhydrous magnesium sulfate is 80 mg;
  • the content of formic acid in the mobile phase B is 0.1%
  • the content of methanol in the complex solution is 100%.
  • its use method includes the following steps:
  • Sample detection Inject 5 ⁇ L each of the processed calibrator working solution, quality control working solution, and urine sample into a high-performance liquid chromatography-tandem mass spectrometer for detection and analysis, and record the chromatogram and sora in the detected sample.
  • the chromatographic conditions in step (5) are:
  • the mass spectrometry conditions are:
  • Ion transfer tube temperature 350°C
  • the Q1/Q3 ion channels are respectively selected as
  • Sorafenib 465.1 ⁇ 252.1, 270.1amu;
  • This kit already contains the main supporting reagents and consumables required for tandem mass spectrometry detection of TKIs in human urine, and simplifies the experimental operation steps, thus making clinical testing easier and faster, greatly reducing the separate purchase and self-service of reagents and consumables Operational errors caused by preparing reagents.
  • the detection method used in this kit is high-performance liquid chromatography tandem mass spectrometry, which directly detects sorafenib, lenvatinib, regorafenib, and cabozantin in urine based on the inherent physical and chemical properties of the analytes.
  • the advantage of high specificity is that it can accurately quantify the status of each analyte in the body.
  • This kit uses the QuEChERS method for pretreatment, using acetonitrile as the extraction agent, sodium chloride and anhydrous magnesium sulfate as the salting out agent, and C18 as the adsorbent. Compared with traditional pretreatment methods, it can effectively remove clinical urine The matrix effect improves the purification efficiency, thereby more clearly reflecting the concentration of therapeutic drugs in the patient's body.
  • Figure 1 is a flow chart of sample processing operations of the present invention
  • Figure 2 is the standard curve of lenvatinib in the embodiment
  • Figure 3 is the standard curve of cabozantinib in the embodiment
  • Figure 4 is the standard curve of Galunisertib in the Example
  • Figure 5 is the standard curve of regorafenib in the embodiment
  • Figure 6 is the standard curve of sorafenib in the embodiment
  • Figure 7 is the standard curve of apatinib in the embodiment.
  • Figure 8 is the standard curve of tivantinib in the embodiment
  • Figure 9 is the standard curve of gefitinib in the embodiment.
  • Figure 10 is the standard curve of anlotinib in the embodiment
  • Figure 11 is the chromatogram of lenvatinib in the embodiment.
  • Figure 12 is the chromatogram of cabozantinib in the embodiment.
  • Figure 13 is the chromatogram of Galunisertib in the Example
  • Figure 14 is the chromatogram of regorafenib in the embodiment.
  • Figure 15 is the chromatogram of sorafenib in the embodiment.
  • Figure 16 is the chromatogram of apatinib in the embodiment.
  • Figure 17 is the chromatogram of tivantinib in the embodiment.
  • Figure 18 is the chromatogram of gefitinib in the embodiment.
  • Figure 19 is the chromatogram of anlotinib in the embodiment.
  • Figure 20 is a chromatogram of propranolol in the Example.
  • the invention is a kit for the detection of TKIs in human urine by tandem mass spectrometry, which provides a mature kit and corresponding operating methods for clinical detection.
  • kit components and operating methods are included, as follows:
  • Calibration materials A, B, C, D, E, F, G
  • quality control materials C1, C2, C3
  • internal standard stock solutions At room temperature, accurately measure 1000 ⁇ L of methanol into each bottle In calibrator or quality control product, completely dissolve and set aside.
  • (2) Working solution Add methanol to continue diluting the calibrator, quality control and internal standard stock solutions in step (1) to the required concentration.
  • the final concentration of the calibrator working solution is: 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 80ng/mL, 100ng/mL (tivantinib, Galunisrtib); 0.2ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 8ng/mL, 10ng/mL (sorafenib, lenvatinib, regorafenib, cabozantinib, anlotinib, apatinib and gefitinib).
  • the final concentrations of quality control working solutions are: 2ng/mL, 50ng/mL, 100ng/mL (tivantinib and galunisrtib); 0.2ng/mL, 5ng/mL, 10ng/mL (sorafenib, lenvatinib) regorafenib, cabozantinib, anlotinib, apatinib and gefitinib).
  • the final concentration of the internal standard working solution is 5ng/mL.
  • Mobile phase mobile phase A liquid, mobile phase B liquid;
  • Ion source electrospray ionization (ESI);
  • the ion source parameters are as follows:
  • Ion source parameters may vary depending on the actual instrument model.
  • the mass spectrum parameters are as follows:
  • Mass spectrometry voltage parameters may vary depending on the actual instrument model.
  • Standard curve drawing method use the labeled concentrations of 7 calibrators (tivantinib, Galunisrtib: 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 80ng/mL, 100ng /mL; sorafenib, lenvatinib, regorafenib, cabozantinib, anlotinib, apatinib, gefitinib: 0.2ng/mL, 0.5ng/mL, 1ng/ mL, 2ng/mL, 5ng/mL, 8ng/mL, 10ng/mL) as the abscissa (x), and the ratio of the actual detected peak areas of the seven calibrators to their respective internal standard peak areas as the ordinate (y), Draw a standard curve.
  • 7 calibrators tivantinib, Galunisrtib: 2ng/mL, 5ng/mL, 10ng/mL, 20ng/m

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Abstract

涉及抑制剂检测技术领域，一种人体尿液抗肝癌酪氨酸激酶抑制剂串联质谱检测试剂盒，包括校准品、质控品、内标准液、提取剂、QuEChERS净化材料、盐析剂、流动相A液、流动相B液、复溶液、隔水包装袋及其分隔并集中包装这些试剂瓶或管的包装盒。将QuEChERS技术和液质联用技术相结合，样品用量少，且结果准确、可靠、灵敏，适合同时检测多种具有抗肝癌作用的TKIs；而且试剂盒具备操作简便、容易推广的优点，可以避免自配试剂过程中引起的实验误差，从而提高临床检测质量。

Description

[根据细则26改正 03.03.2023]一种人体尿液抗肝癌酪氨酸激酶抑制剂串联质谱检测试剂盒 技术领域
本发明属于抑制剂检测技术领域，具体是一种人体尿液抗肝癌酪氨酸激酶抑制剂串联质谱检测试剂盒。
背景技术
酪氨酸激酶抑制剂(tyrosine kinase inhibitor，TKIs)的作用机制是与三磷酸腺苷(ATP)竞争激酶域的结合位点并降低酪氨酸激酶的磷酸化，从而抑制癌细胞增殖。TKIs与传统的细胞毒类抗癌药比较，具有高选择性，不良反应少等特点，广泛用于小细胞肺癌，非小细胞肺癌，胃肠间质瘤，肝细胞肝癌，肾癌等癌症。目前，TKIs在癌症治疗方面取得了很大进展，TKIs与肝动脉化疗栓塞术的联合治疗相比于单一肝动脉栓塞化疗术效果更好，能有效降低不良反应的发生率。
尿液是小分子代谢物研究中常用的生物液体，样品复杂性低，前处理相对简单，且尿液中代谢物丰富，在进行分析前只需准备少量样本，采集简单且无创。因此，本发明针对人体尿液样本建立同时检测肝癌患者尿液中9种TLIs的方法，较全面的涵盖了当前具有抗肝癌作用的TKIs，对临床治疗药物监测具有重要意义。
目前，国内外文献中报道了多种分析方法用于检测小分子酪氨酸激酶抑制剂，主要包括高效液相色谱法(high performance liquid chromatography，HPLC)、薄层色谱法(thin layer chromatography，TLC)、酶联免疫分析法(enzyme-linked immuno sorbent assay，ELISA)、气相色谱串联质谱法(Gas chromatography-mass spectrometry，GC-MS/MS)和液相色谱串联质谱法(liquid chromatography-mass spectrometry，LC-MS/MS)等。其中，LC-MS/MS结合了色谱的分离能力和质谱的分析能力，且具有动态范围大、分析速度快、 灵敏度高、检出限低等优点，是小分子代谢物分析的关键技术。同时，检测时需要样品有良好的基质效应，因此需要选择合适的前处理技术，以提高净化效率，减小杂质影响。QuEChERS是由分散固相萃取(dSPE)衍生出的一种新兴的前处理技术，具有回收率高，操作简单等优点，最初用于农药残留领域，近年来，随着该技术的改良和发展，逐渐应用于尿液、血浆和组织等生物基质中代谢物和其他化合物的分析。
肝细胞癌(hepatocellular carcinoma，HCC)是一种预后不佳的疾病，具有发病率高，死亡率高和早期不易发现的特点。本发明同时检测的9种酪氨酸激酶抑制剂，都是具有抗肝癌作用的小分子靶向药物。尽管分子靶向治疗在晚期肝癌的治疗中发挥着不可替代的作用，但获得耐药性和药物毒性仍然不可避免，因此也限制了癌症的治疗。于是需要对治疗药物进行监测，辅助临床用药指导，及时调整给药剂量，提高药物疗效和安全性。
因此，临床检测亟需一种不仅具备高灵敏度，高特异性，高准确度检测结果，还可以同时检测多种具有抗肝癌作用的TKIs，并拥有操作简单方便，容易推广的TKIs直接检测方法及试剂盒，本发明通过将QuEChERS前处理方法与高效液相色谱串联质谱联用，来减小基质效应和化合物不稳定性对实验结果的影响，从而提高临床检测质量。
发明内容
本发明的目的是针对以上问题，提供了一种人体尿液抗肝癌酪氨酸激酶抑制剂串联质谱检测试剂盒，其特征在于，包括校准品、质控品、内标准液、提取剂、QuEChERS净化材料、盐析剂、流动相A液、流动相B液、复溶液、隔水包装袋及其分隔并集中包装这些试剂瓶或管的包装盒。
优选的，校准品：使用甲醇稀释液，同时加入替万替尼、Galunisrtib、索拉非尼，伦伐替尼，瑞戈非尼，卡博替尼，安罗替尼，阿帕替尼和吉非替尼的标准品，配制成7个浓度剂量点的校准品A-G，其中替万替尼和 Galunisrtib的浓度分别为2ng/mL、5ng/mL、1Ong/mL、20ng/mL、50ng/mL、80ng/mL、100ng/mL，索拉非尼、伦伐替尼、瑞戈非尼、卡博替尼、安罗替尼、阿帕替尼和吉非替尼的浓度分别为0.2ng/mL、0.5ng/mL、1ng/mL、2ng/mL、5ng/mL、8ng/mL、10ng/mL；
质控品：使用甲醇稀释液，同时加入替万替尼、Galunisrtib、索拉非尼，伦伐替尼，瑞戈非尼，卡博替尼，安罗替尼，阿帕替尼和吉非替尼的标准品，配制成3个浓度的质控品C1-C3，其中替万替尼和Galunisrtib的浓度分别为2ng/mL、50ng/mL、100ng/mL，索拉非尼、伦伐替尼、瑞戈非尼、卡博替尼、安罗替尼、阿帕替尼和吉非替尼的浓度分别为0.2ng/mL、5ng/mL、10ng/mL；
内标准液：使用甲醇作为稀释液，将普萘洛尔稀释至终浓度为5ng/mL；
提取剂：1-3mL乙腈；
QuEChERS净化材料：30-60mg C18，用隔水包装袋封装；
盐析剂：10-40mg氯化钠、50-100mg无水硫酸镁，用隔水包装袋封装；
流动相A：为纯乙腈；
流动相B：由甲酸与纯化水配制而成，甲酸的含量为0.05％-0.2％；
复溶液：由甲醇和纯化水配制而成，甲醇的含量为50％-100％。
优选的，所述提取剂中尿液：乙腈＝1：2，乙腈的用量为2mL；所述C18的用量为50mg；
所述盐析剂中氯化钠的用量为20mg，无水硫酸镁的用量为80mg；
所述流动相B中甲酸的含量为0.1％；
所述复溶液中甲醇的含量为100％。
优选的，其使用方法包括以下步骤：
(1)准备工作液：用甲醇分别稀释试剂盒中的校准品、质控品、内标准液，分别制成校准品工作液、质控品工作液、内标工作液；
(2)样品前处理：取1mL的尿液样品与10μL的内标工作液混合，依次加入提取剂、QuEChERS净化材料和盐析剂，混合均匀后离心分离上清，氮气室温彻底吹干，加入1mL复溶液至溶解完全；
(3)混匀：将步骤(2)中得到的溶液用涡旋混匀器充分混合1min；
(4)转移：取复溶后的溶液200μL转移至液相小瓶，待测。
(5)设置色谱和质谱条件：根据实际仪器型号分别设置超高效液相色谱和串联质谱仪的工作参数和条件；
(6)样品检测：将处理好的校准品工作液、质控品工作液和尿液样品各5μL注入高超效液相色谱-串联质谱仪进行检测分析，并记录色谱图和检测样品中索拉非尼，伦伐替尼，瑞戈非尼，卡博替尼，安罗替尼，阿帕替尼，吉非替尼，替万替尼和Galunisrtib的峰面积与内标峰面积；
(6)结果分析：绘制标准曲线和计算曲线回归方程，分别计算出样品中索拉非尼，伦伐替尼，瑞戈非尼，卡博替尼，安罗替尼，阿帕替尼，吉非替尼，替万替尼和Galunisrtib的浓度。
优选的，所述步骤(1)中，内标工作液按照按内标准液∶甲醇＝1∶99配制而成。
优选的，所述步骤(5)中的色谱条件为：
色谱柱：Hypersil GOLD VANQUISH C18(2.1×100mm,1.9μm)，
流速：0.3mL/min，
柱温：40℃，
进样量：5μL，
梯度洗脱条件：
Figure PCTCN2022139272-appb-000001
Figure PCTCN2022139272-appb-000002
质谱条件为：
ESI正离子SRM扫描模式，
喷雾电压：3500V，
鞘气流速：45Arb，
辅助气流速：10Arb，
离子传输管温度：350℃，
雾化温度：400℃，
碰撞气：氩气，
压力：1.5mTor；
Q1/Q3离子通道分别选择为
伦伐替尼:427.1→370.1，312.0amu；
索拉非尼:465.1→252.1，270.1amu；
卡博替尼:502.2→323.1，297.1amu；
瑞戈非尼:483.1→270.1，288.1amu；
阿帕替尼:398.2→212.1，184.1amu；
吉非替尼:447.1→128.1，100.1amu；
安罗替尼:408.2→339.1，304.1amu；
替万替尼:370.1→253.1，158.1amu；
Galunisertib:370.1→336.1，325.1amu。
与现有技术相比，本发明的有益效果如下：
1、本试剂盒已经含有人体尿液中TKIs检测串联质谱法所需的主要配套试剂和耗材，并且简化了实验操作步骤，从而能使临床检测更加简便快捷，大大减少试剂耗材的单独购买和自行配制试剂带来的操作误差。
2、本试剂盒使用的检测方法为高效液相色谱串联质谱法，根据分析物固有的理化性质进行直接检测尿液中的索拉非尼，伦伐替尼，瑞戈非尼，卡博替尼，安罗替尼，阿帕替尼，吉非替尼，替万替尼和Galunisrtib 9种TKIs，具有高灵敏度。高特异性的优点，能够准确的定量出各个分析物质在体内的情况。
3、本试剂盒采用QuEChERS方法进行前处理，采用乙腈作为提取剂，氯化钠和无水硫酸镁作为盐析剂，C18作为吸附剂，与传统前处理方法相比，能够有效去除临床尿液的基质效应，提高净化效率，从而更加清楚的反映患者体内治疗药物的浓度。
附图说明
图1为本发明的样品处理操作流程图；
图2为实施例中伦伐替尼的标准曲线；
图3为实施例中卡博替尼的标准曲线；
图4为实施例中Galunisertib的标准曲线；
图5为实施例中瑞戈非尼的标准曲线；
图6为实施例中索拉非尼的标准曲线；
图7为实施例中阿帕替尼的标准曲线；
图8为实施例中替万替尼的标准曲线；
图9为实施例中吉非替尼的标准曲线；
图10为实施例中安罗替尼的标准曲线；
图11为实施例中伦伐替尼的色谱图；
图12为实施例中卡博替尼的色谱图；
图13为实施例中Galunisertib的色谱图；
图14为实施例中瑞戈非尼的色谱图；
图15为实施例中索拉非尼的色谱图；
图16为实施例中阿帕替尼的色谱图；
图17为实施例中替万替尼的色谱图；
图18为实施例中吉非替尼的色谱图；
图19为实施例中安罗替尼的色谱图；
图20为实施例中普萘洛尔的色谱图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明为一种人体尿液中TKIs串联质谱检测试剂盒，为临床检测提供了配套成熟的试剂盒以及所包含对应的操作方法。为了使本发明实施的上述目的、特征和优点能够更加清晰易懂，包括试剂盒组分和操作方法，具体如下：
试剂盒组分
Figure PCTCN2022139272-appb-000003
Figure PCTCN2022139272-appb-000004
试剂盒配套操作方法
1.试剂组分配制：
(1)校准品(A、B、C、D、E、F、G)和质控品(C1、C2、C3)和内标准品储备液：室温条件下，准确量取1000μL甲醇至每瓶校准品或质控品中，完全溶解，备用。
(2)工作液：加入甲醇继续稀释步骤(1)的校准品、质控品和内标准品储 备液，至所需浓度，其中校准品工作液最终浓度为：2ng/mL、5ng/mL、10ng/mL、20ng/mL、50ng/mL、80ng/mL、100ng/mL(替万替尼、Galunisrtib)；0.2ng/mL、0.5ng/mL、1ng/mL、2ng/mL、5ng/mL、8ng/mL、10ng/mL(索拉非尼，伦伐替尼，瑞戈非尼，卡博替尼，安罗替尼，阿帕替尼和吉非替尼)。质控品工作液最终浓度为：2ng/mL、50ng/mL、100ng/mL(替万替尼和Galunisrtib)；0.2ng/mL、5ng/mL、10ng/mL(索拉非尼，伦伐替尼，瑞戈非尼，卡博替尼，安罗替尼，阿帕替尼和吉非替尼)。内标工作溶液的最终浓度为5ng/mL。
2.检测样品处理：参见图1。
a)内标混合：取1mL的尿液样品与10μL的内标准品工作液充分混合；
b)前处理：依次准确加入提取剂、净化材料和盐析剂，混合均匀后以13000rpm离心10min分离上清；
c)氮气吹干：离心后取上清液至离心管中用氮气在室温下彻底吹干；
d)复溶：然后各加入1mL样品复溶液，混合均匀；
d)转移：复溶后转移200μL至液相小瓶，准备上机进样分析。
3.色谱条件：
a)色谱柱：Hypersil GOLD VANQUISH C18(2.1×100mm,1.9μm)或相当者；
b)流动相：流动相A液，流动相B液；
c)流速：0.3mL/min；
d)梯度洗脱条件：
Figure PCTCN2022139272-appb-000005
Figure PCTCN2022139272-appb-000006
e)柱温：40℃；
f)进样量：5μL。
4.质谱条件：
a)离子源：电喷雾电离(ESI)；
b)扫描模式：正离子SRM扫描分析，Q1/Q3离子通道分别选择为伦伐替尼:427.1→370.1，312.0amu；索拉非尼:465.1→252.1，270.1amu；卡博替尼:502.2→323.1，297.1amu；瑞戈非尼:483.1→270.1，288.1amu；阿帕替尼:398.2→212.1，184.1amu；吉非替尼:447.1→128.1，100.1amu；安罗替尼:408.2→339.1，304.1amu；替万替尼:370.1→253.1，158.1amu；Galunisertib:370.1→336.1，325.1amu。
c)本发明实施例的一个优选示例中，离子源参数如下：
Figure PCTCN2022139272-appb-000007
Figure PCTCN2022139272-appb-000008
离子源参数可根据实际仪器型号不同而有所不同。
a)本发明实施例的一个优选示例中，质谱参数如下：
Figure PCTCN2022139272-appb-000009
a定量离子
质谱电压参数可根据实际仪器型号不同而有所不同。
5.样品检测：
选取处理好的校准品、质控品和尿液样品各5μL注入超高效液相色谱-串联质谱仪进行检测分析，并记录色谱图与检测样品中索拉非尼，伦伐替尼，瑞戈非尼，卡博替尼，安罗替尼，阿帕替尼，吉非替尼，替万替尼和Galunisrtib的峰面积与内标峰面积。
6.检测结果的计算：
(1)标准曲线的绘制方法：以7个校准品的标示浓度(替万替尼、Galunisrtib： 2ng/mL、5ng/mL、10ng/mL、20ng/mL、50ng/mL、80ng/mL、100ng/mL；索拉非尼、伦伐替尼、瑞戈非尼、卡博替尼、安罗替尼、阿帕替尼、吉非替尼：0.2ng/mL、0.5ng/mL、1ng/mL、2ng/mL、5ng/mL、8ng/mL、10ng/mL)为横坐标(x)，以7个校准品的实际检测峰面积与各自内标峰面积的比值为纵坐标(y)，绘制标准曲线。
(2)标准曲线方程的拟合：以7个校准品的峰面积比值(y)对标示浓度(x)进行线性回归。可获得回归方程：y＝a+bx，其中y为纵坐标，x为横坐标，a为截距，b为斜率，并计算相关系数(r)，要求r不小于0.9900。
(3)尿液样品结果的计算：将尿液样品中TKIs的实际检测峰面积与内标峰面积的比值代入标准曲线方程，计算尿液样品中TKIs的浓度。
实施例：健康人尿液中9种酪氨酸激酶抑制剂的定量分析
1、线性实验
取空白尿液样品，按图1所示流程进行操作，按上述“检测结果的计算”部分进行结果分析，得到各分析物和内标的线性回归方程(如图2-10所示)。所有分析物的线性相关系数(R 2)在0.9975～0.9993之间。
2、专属性考察
在健康人的尿液中加入9种酪氨酸激酶抑制剂，按图1所示流程进行分析，其中，9种酪氨酸激酶抑制剂和一种内标在SRM模式下的典型色谱图如图11-20所示。实验结果显示，9种物质分离效果较好，且每个分析物的峰型良好，表明该方法具有较好的选择性，可以用于尿液中9种酪氨酸激酶抑制剂的定量检测。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系 列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (6)

  1. 一种人体尿液抗肝癌酪氨酸激酶抑制剂串联质谱检测试剂盒，其特征在于，包括校准品、质控品、内标准液、提取剂、QuEChERS净化材料、盐析剂、流动相A液、流动相B液、复溶液、隔水包装袋及其分隔并集中包装这些试剂瓶或管的包装盒；
    QuEChERS净化材料：30-60mg C18，用隔水包装袋封装；
    盐析剂：10-40mg氯化钠、50-100mg无水硫酸镁，用隔水包装袋封装。
  2. 根据权利要求1所述的一种人体尿液抗肝癌酪氨酸激酶抑制剂串联质谱检测试剂盒，其特征在于，
    校准品：使用甲醇稀释液，同时加入替万替尼、Galunisrtib、索拉非尼，伦伐替尼，瑞戈非尼，卡博替尼，安罗替尼，阿帕替尼和吉非替尼的标准品，配制成7个浓度剂量点的校准品A-G，其中替万替尼和Galunisrtib的浓度分别为2ng/mL、5ng/mL、10ng/mL、20ng/mL、50ng/mL、80ng/mL、100ng/mL，索拉非尼、伦伐替尼、瑞戈非尼、卡博替尼、安罗替尼、阿帕替尼和吉非替尼的浓度分别为0.2ng/mL、0.5ng/mL、1ng/mL、2ng/mL、5ng/mL、8ng/mL、10ng/mL；
    质控品：使用甲醇稀释液，同时加入替万替尼、Galunisrtib、索拉非尼，伦伐替尼，瑞戈非尼，卡博替尼，安罗替尼，阿帕替尼和吉非替尼的标准品，配制成3个浓度的质控品C1-C3，其中替万替尼和Galunisrtib的浓度分别为2ng/mL、50ng/mL、100ng/mL，索拉非尼、伦伐替尼、瑞戈非尼、卡博替尼、安罗替尼、阿帕替尼和吉非替尼的浓度分别为0.2ng/mL、5ng/mL、10ng/mL；
    内标准液：使用甲醇作为稀释液，将普萘洛尔稀释至终浓度为5ng/mL；
    提取剂：1-3mL乙腈；
    流动相A：为纯乙腈；
    流动相B：由甲酸与纯化水配制而成，甲酸的含量为0.05％-0.2％；
    复溶液：由甲醇和纯化水配制而成，甲醇的含量为50％-100％。
  3. 根据权利要求1或2任一所述的一种人体尿液抗肝癌酪氨酸激酶抑制剂串联质谱检测试剂盒，其特征在于，
    所述提取剂中尿液：乙腈＝1：2，乙腈的用量为2mL；
    所述C18的用量为50mg；
    所述盐析剂中氯化钠的用量为20mg，无水硫酸镁的用量为80mg；
    所述流动相B中甲酸的含量为0.1％；
    所述复溶液中甲醇的含量为100％。
  4. 根据权利要求1所述的一种人体尿液抗肝癌酪氨酸激酶抑制剂串联质谱检测试剂盒，其特征在于，其使用方法包括以下步骤：
    (1)准备工作液：用甲醇分别稀释试剂盒中的校准品、质控品、内标准液，分别制成校准品工作液、质控品工作液、内标工作液；
    (2)样品前处理：取1mL的尿液样品与10μL的内标工作液混合，依次加入提取剂、QuEChERS净化材料和盐析剂，混合均匀后离心分离上清，氮气室温彻底吹干，加入1mL复溶液至溶解完全；
    (3)混匀：将步骤(2)中得到的溶液用涡旋混匀器充分混合1min；
    (4)转移：取复溶后的溶液200μL转移至液相小瓶中，待测；
    (5)设置色谱和质谱条件：根据实际仪器型号分别设置超高效液相色谱和串联质谱仪的工作参数和条件；
    (6)样品检测：将处理好的校准品工作液、质控品工作液和尿液样品各5μL注入高超效液相色谱-串联质谱仪进行检测分析，并记录色谱图和检测样品中索拉非尼，伦伐替尼，瑞戈非尼，卡博替尼，安罗替尼，阿帕替尼，吉非替尼，替万替尼和Galunisrtib的峰面积与内标峰面积；
    (7)结果分析：绘制标准曲线和计算曲线回归方程，分别计算出样品中索拉非尼，伦伐替尼，瑞戈非尼，卡博替尼，安罗替尼，阿帕替尼，吉非替 尼，替万替尼和Galunisrtib的浓度。
  5. 根据权利要求4所述的一种人体尿液抗肝癌酪氨酸激酶抑制剂串联质谱检测试剂盒，其特征在于，所述步骤(1)中，内标工作液按照按内标准液∶甲醇＝1∶99配制而成。
  6. 根据权利要求4所述的一种人体尿液抗肝癌酪氨酸激酶抑制剂串联质谱检测试剂盒，其特征在于，所述步骤(5)中的色谱条件为：
    色谱柱：Hypersil GOLD VANQUISH C18(2.1×100mm,1.9μm)，
    流速：0.3mL/min，
    柱温：40℃，
    进样量：5μL，
    梯度洗脱条件：
    Figure PCTCN2022139272-appb-100001
    质谱条件为：
    ESI正离子SRM扫描模式，
    喷雾电压：3500V，
    鞘气流速：45Arb，
    辅助气流速：10Arb，
    离子传输管温度：350℃，
    雾化温度：400℃，
    碰撞气：氩气，
    压力：1.5mTor；
    Q1/Q3离子通道分别选择为
    伦伐替尼:427.1→370.1，312.0amu；
    索拉非尼:465.1→252.1，270.1amu；
    卡博替尼:502.2→323.1，297.1amu；
    瑞戈非尼:483.1→270.1，288.1amu；
    阿帕替尼:398.2→212.1，184.1amu；
    吉非替尼:447.1→128.1，100.1amu；
    安罗替尼:408.2→339.1，304.1amu；
    替万替尼:370.1→253.1，158.1amu；
    Galunisertib:370.1→336.1，325.1amu。
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