CN115541778B - A kind of detection method for measuring the concentration of apremilast in human plasma - Google Patents
A kind of detection method for measuring the concentration of apremilast in human plasma Download PDFInfo
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Abstract
本发明涉及药物分析技术领域,尤其涉及一种测定人血浆中阿普斯特浓度的检测方法。所述血浆中阿普斯特浓度的检测方法,包括以下步骤:将含阿普斯特的血浆、阿普斯特‑d5的乙腈溶液混合进行沉淀反应,将所得上清液分别注入液相色谱‑质谱仪,进行多反应监测反应,用内标法以标准曲线计算血浆中阿普斯特的含量,所述标准曲线以阿普斯特的浓度为横坐标,以阿普斯特和阿普斯特‑d5的峰面积比值为纵坐标。本发明基于沉淀法和液质联用技术进行血浆中阿普斯特的检测,可用于检测生物体内的阿普斯特浓度,提供了一种快速、准确、稳定的检测方法。
The invention relates to the technical field of drug analysis, in particular to a detection method for measuring the concentration of apremilast in human blood plasma. The detection method of apremilil concentration in the plasma comprises the following steps: mixing the acetonitrile solution containing the plasma of apremilast and apremilast-d5 to carry out the precipitation reaction, and injecting the supernatant of gained into liquid chromatography respectively ‑Mass spectrometer, carry out multiple reaction monitoring reaction, use internal standard method to calculate the content of Apremilast in plasma with standard curve, described standard curve takes the concentration of Apremilast as abscissa, takes Apremilast and Apremilast The ratio of the peak areas of ster-d5 is the ordinate. The invention detects the apremilast in the blood plasma based on the precipitation method and liquid mass spectrometry technology, can be used to detect the concentration of the apremilast in the living body, and provides a fast, accurate and stable detection method.
Description
技术领域technical field
本发明涉及药物分析技术领域,尤其涉及一种血浆中阿普斯特浓度的检测方法。The invention relates to the technical field of drug analysis, in particular to a method for detecting the concentration of apremilast in plasma.
背景技术Background technique
阿普斯特是一种口服的环单磷酸腺苷(cAMP)特异性磷酸二酯酶-4(PDE4)小分子抑制剂。PDE4的抑制能够升高细胞内的cAMP水平,PDE4降解cAMP可导致免疫细胞激活和促炎细胞因子释放。因此抑制PDE4可能能够降低PDE4介导的炎症反应。阿普斯特片,2014年03月21日在美国获批上市销售,批准的适应症为活动性银屑病关节炎,适用光疗或者系统治疗的中度至重度斑块状银屑病和与白塞病相关的口腔溃疡。2014年9月,FDA 批准阿普斯特用于治疗中重度斑块状银屑病。由于其有希望的治疗结果和安全性,该药物分子在皮肤病学中变得非常流行。据研究报道,目前国内有关测定人体内阿普斯特血药浓度的方法尚未研究,而测定其在生物体内的血浆药物浓度对于评价药物疗效和分析药理作用等方面具有很重要的作用,因此开发一种测定血浆中阿普斯特浓度的检测方法,进行阿普斯特在中国健康受试者空腹及餐后状态下体内的药动学及人体生物等效性研究,不仅可以为阿普斯特相关制剂一致性评价提供依据,而且具有广泛的经济效益和社会效益。Apremilast is an oral, small-molecule inhibitor of cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase-4 (PDE4). Inhibition of PDE4 increases intracellular cAMP levels, and degradation of cAMP by PDE4 leads to immune cell activation and release of pro-inflammatory cytokines. Therefore, inhibition of PDE4 may be able to reduce PDE4-mediated inflammatory response. Apremilast tablets were approved for marketing in the United States on March 21, 2014. The approved indications are active psoriatic arthritis, moderate to severe plaque psoriasis suitable for phototherapy or systemic treatment and related Oral ulcers associated with Behcet's disease. In September 2014, the FDA approved apremilast for the treatment of moderate to severe plaque psoriasis. This drug molecule has become very popular in dermatology due to its promising therapeutic results and safety profile. According to research reports, at present, domestic methods for determining the blood concentration of apremilast in the human body have not yet been studied, and the determination of its plasma drug concentration in the living body plays a very important role in evaluating the efficacy of the drug and analyzing the pharmacological effects. Therefore, the development of A detection method for determining the concentration of apremilast in plasma, the pharmacokinetics and human bioequivalence studies of apremilast in Chinese healthy subjects under fasting and postprandial conditions, can not only provide It provides a basis for the consistency evaluation of special related preparations, and has extensive economic and social benefits.
发明内容Contents of the invention
为解决现有技术存在的技术问题,本发明的目的在于提供一种血浆中阿普斯特浓度的检测方法。本发明基于沉淀法和液质联用技术进行血浆中阿普斯特的检测,可用于检测生物体内的阿普斯特浓度,提供了一种快速、准确、稳定的检测方法。In order to solve the technical problems in the prior art, the object of the present invention is to provide a method for detecting the concentration of apremilast in plasma. The invention detects the apremilast in the blood plasma based on the precipitation method and liquid mass spectrometry technology, can be used to detect the concentration of the apremilast in the living body, and provides a fast, accurate and stable detection method.
为了实现上述发明目的,本发明提供以下技术方案:In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:
本发明提供了一种血浆中阿普斯特浓度的检测方法,包括以下步骤:The invention provides a method for detecting the concentration of apremilast in plasma, comprising the following steps:
将含阿普斯特的血浆、阿普斯特-d5的乙腈溶液混合进行沉淀反应,涡旋混合5min,置4 ℃台式高速冷冻离心机(96孔板)中,4750 r/min离心10 min,每孔取上清100 µL,转移至另一96孔板,每孔加10%乙腈水溶液100 µL稀释,涡旋混合5 min后,进行液质联用检测,用内标法以标准曲线计算血浆中阿普斯特的含量,所述标准曲线以阿普斯特的浓度为横坐标,以阿普斯特和阿普斯特-d5的峰面积比值为纵坐标。The plasma containing apremilast and the acetonitrile solution of apremilast-d5 were mixed for precipitation reaction, vortexed for 5 min, placed in a desktop high-speed refrigerated centrifuge (96-well plate) at 4 °C, and centrifuged at 4750 r/min for 10 min , take 100 µL of the supernatant from each well, transfer to another 96-well plate, add 100 µL of 10% acetonitrile aqueous solution to each well to dilute, vortex and mix for 5 min, perform liquid chromatography-mass spectrometry detection, and use the internal standard method to calculate the standard curve The content of apremilil in plasma, the standard curve takes the concentration of apremilast as the abscissa, and the peak area ratio of apremilil and apremilil-d5 as the ordinate.
进一步地,所述液质联用检测的高效液相色谱条件包括:色谱柱为PhenomenexKinetex C18 (2.1×50 mm,2.6 µm)色谱柱,采用梯度洗脱,流动相A为含0.1%甲酸的水溶液,流动相B为0.1 %甲酸乙腈溶液,洗脱程序为0~0.5 min,流动相A的体积百分含量保持63%,流动相B的体积含量保持37%;0.5~1.3 min,流动相A的体积百分含量从63%下降到0.0%,流动相B的体积百分含量从37%上升到100%;1.3~2.4 min,流动相A的体积百分含量保持0.0%,流动相B的体积含量保持100%;2.4~2.41 min,流动相A的体积百分含量从0.0%上升到63%,流动相B的体积百分含量从100%下降到37%;2.41~3 min,流动相A的体积百分含量保持63%,流动相B的体积含量保持37%,流速为0.45 mL/min,柱温为40℃,进样量为5 µL;Further, the high-performance liquid chromatography conditions detected by liquid chromatography-mass spectrometry include: the chromatographic column is a Phenomenex Kinetex C18 (2.1×50 mm, 2.6 µm) chromatographic column, gradient elution is used, and the mobile phase A is an aqueous solution containing 0.1% formic acid , the mobile phase B is 0.1% formic acid acetonitrile solution, the elution program is 0~0.5 min, the volume percentage of mobile phase A is kept at 63%, and the volume content of mobile phase B is kept at 37%; 0.5~1.3 min, mobile phase A The volume percentage of mobile phase A decreased from 63% to 0.0%, and the volume percentage of mobile phase B increased from 37% to 100%; 1.3~2.4 min, the volume percentage of mobile phase A remained at 0.0%, and the volume percentage of mobile phase B The volume content remained at 100%; 2.4~2.41 min, the volume percentage of mobile phase A increased from 0.0% to 63%, and the volume percentage of mobile phase B decreased from 100% to 37%; 2.41~3 min, the mobile phase The volume percentage of A was kept at 63%, the volume content of mobile phase B was kept at 37%, the flow rate was 0.45 mL/min, the column temperature was 40°C, and the injection volume was 5 µL;
进一步地,所述液质联用检测的质谱条件包括:使用三重四级杆质谱仪,离子源为电喷雾离子源,采用负离子模式,采用多反应监测扫描模式,离子化电压:5500 V;入口电压(EP):10 V;碰撞室出口电压(CXP):13 V;喷雾气(Gas1):50;辅助加热气(Gas2):55;气帘气:35,所述阿普斯特的离子对为461.2/257.1(定量离子对),碰撞能量(CE):14 V,去簇电压(DP):190 V;所述阿普斯特-d5的离子对为466.1/262.3,CE:14 V,DP:190 V。Further, the mass spectrometry conditions detected by liquid chromatography-mass spectrometry include: using a triple quadrupole mass spectrometer, the ion source is an electrospray ion source, using negative ion mode, using multiple reaction monitoring scanning mode, ionization voltage: 5500 V; inlet Voltage (EP): 10 V; Collision Cell Exit Voltage (CXP): 13 V; Spray Gas (Gas1): 50; Auxiliary Heating Gas (Gas2): 55; is 461.2/257.1 (quantitative ion pair), collision energy (CE): 14 V, declustering voltage (DP): 190 V; the ion pair of Apremilast-d5 is 466.1/262.3, CE: 14 V, DP: 190V.
进一步地,含阿普斯特的血浆样品检测方法为:取96孔板,每孔加含阿普斯特的血浆样品100 µL,再加质量浓度为20.0 ng/mL内标工作溶液(内标工作溶液稀释剂为乙腈溶液))300 µL,涡旋混合5 min,置4 ℃台式高速冷冻离心机(96孔板)中,4750 r/min离心10min,每孔取上清100 µL,转移至另一96孔板,每孔加10%乙腈水溶液100 µL稀释,涡旋混合5min,进行液质联用检测,用内标法以标准曲线计算血浆中阿普斯特的含量,所述标准曲线以阿普斯特的浓度为横坐标,以阿普斯特和阿普斯特-d5的峰面积比值为纵坐标。Further, the detection method of plasma samples containing apremilast is as follows: take a 96-well plate, add 100 µL of plasma samples containing apremilast to each well, and then add an internal standard working solution with a mass concentration of 20.0 ng/mL (internal standard The working solution diluent is acetonitrile solution)) 300 µL, vortexed for 5 min, placed in a desktop high-speed refrigerated centrifuge (96-well plate) at 4 °C, centrifuged at 4750 r/min for 10 min, and 100 µL of supernatant from each well was transferred to In another 96-well plate, add 100 μL of 10% acetonitrile aqueous solution to each well to dilute, vortex and mix for 5 min, and carry out liquid chromatography-mass spectrometry detection, and use the internal standard method to calculate the content of apremilast in the plasma with the standard curve, the standard curve Take the concentration of apremilast as the abscissa, and the ratio of the peak areas of apremilil and apremilast-d5 as the ordinate.
更进一步地,所述内标工作溶液的制作方法为:称量1 mg阿普斯特-d5标准品,用N,N-二甲基甲酰胺溶解,配成1 mg/mL的内标母液,用乙腈溶液进一步将内标母液稀释为浓度为20.0 ng/mL阿普斯特-d5的乙腈溶液即为内标工作溶液。Further, the preparation method of the internal standard working solution is: weigh 1 mg of Apremilast-d5 standard substance, dissolve it with N,N-dimethylformamide, and prepare a 1 mg/mL internal standard mother solution , using acetonitrile solution to further dilute the internal standard mother solution to acetonitrile solution with a concentration of 20.0 ng/mL Apremilast-d5, which is the internal standard working solution.
进一步地,所述标准曲线由包括以下步骤的方法制得:称量1 mg阿普斯特,用N,N-二甲基甲酰胺溶解得到1mg/mL的阿普斯特母液,精密移取阿普斯特母液适量,用50%乙腈水溶液稀释至质量浓度为100 ng/mL阿普斯特工作溶液,用50%乙腈水溶液将所述工作溶液稀释成40、80、200、600,2000,4000,9600和12000 ng/mL的标准曲线工作液,取空白血浆285µL,加入不同浓度的标准曲线工作液15 µL,得到2.00,4.00,10.0,30.0,100,200,480和600ng/mL的标准曲线样品,取所述标准曲线样品100 µL,加400 µL阿普斯特-d5的乙腈溶液(内标工作溶液)进行沉淀反应,涡旋混合5 min,置4℃台式高速冷冻离心机(96孔板)中,4750r/min离心10 min,每孔取上清100 µL,转移至另一96孔板,每孔加10%乙腈水溶液100 µL稀释,涡旋混合5 min进行液质联用检测,使用内标法对所得数据进行处理,以阿普斯特的浓度为横坐标,阿普斯特和阿普斯特-d5的峰面积比值为纵坐标,以1/x2为权重系数,利用加权最小二乘法进行线性回归,得到所述标准曲线。Further, the standard curve is prepared by a method comprising the following steps: Weigh 1 mg of Apremilast, dissolve it with N,N-dimethylformamide to obtain a 1 mg/mL mother solution of Apremilast, and precisely pipette An appropriate amount of Apremilast mother solution was diluted with 50% acetonitrile aqueous solution to a mass concentration of 100 ng/mL Apremilast working solution, and the working solution was diluted to 40, 80, 200, 600, 2000 with 50% acetonitrile aqueous solution, For standard curve working solution of 4000, 9600 and 12000 ng/mL, take 285 µL of blank plasma and add 15 µL of standard curve working solution of different concentrations to obtain standards of 2.00, 4.00, 10.0, 30.0, 100, 200, 480 and 600 ng/mL For the curve sample, take 100 µL of the standard curve sample, add 400 µL of Apremilast-d5 acetonitrile solution (internal standard working solution) for precipitation reaction, vortex and mix for 5 min, and place in a desktop high-speed refrigerated centrifuge at 4°C (96 orifice plate), centrifuge at 4750r/min for 10 min, take 100 µL of the supernatant from each well, transfer to another 96-well plate, add 100 µL of 10% acetonitrile aqueous solution to each well, vortex and mix for 5 min, and perform liquid chromatography-mass spectrometry detection , using the internal standard method to process the obtained data, with the concentration of Apremilast as the abscissa, the peak area ratio of Apremilast and Apremilast-d5 as the ordinate, and 1/x 2 as the weight coefficient, Linear regression was carried out by weighted least square method to obtain the standard curve.
本发明建立了一种基于沉淀法和液质联用技术的血浆中阿普斯特检测方法,可用于检测生物体内的阿普斯特浓度,为临床和基础研究提供可靠的分析手段,本发明使用蛋白质沉淀法处理含阿普斯特的血浆样品,通过一步沉淀反应去除杂质和内源性干扰物,在高效液相色谱的分离过程中,向流动相中加入了甲酸,使阿普斯特在电离后形成更多的加氢的准分子离子峰,对应的特征性碎片离子的丰度随之升高,提高了检测得灵敏度;在定量的过程中,以阿普斯特-d5为内标物,通过准分子离子峰→子离子的特异性的、高灵敏度的监测实现对阿普斯特的定量检测。The present invention establishes a method for detecting apremilast in plasma based on precipitation method and liquid chromatography-mass spectrometry technology, which can be used to detect the concentration of apremilast in living organisms and provide reliable analysis means for clinical and basic research. The present invention Use the protein precipitation method to process the plasma samples containing apremilast, and remove impurities and endogenous interference substances through a one-step precipitation reaction. During the separation process of high performance liquid chromatography, formic acid is added to the mobile phase to make apremilast After ionization, more hydrogenated quasi-molecular ion peaks are formed, and the abundance of corresponding characteristic fragment ions increases accordingly, which improves the detection sensitivity; in the quantitative process, Apremilast-d5 is used as the internal The quantitative detection of apremilast is realized through the specific and high-sensitivity monitoring of quasi-molecular ion peak→product ion.
本发明的有益效果Beneficial effects of the present invention
本发明建立的测定人血浆中阿普斯特浓度的方法具有灵敏度高、准确可靠、选择性好、基质效应小、快速简便等优点。该方法使用乙腈对血浆样品进行蛋白沉淀,内标工作溶液直接配制到沉淀剂中,减少了操作步骤,使操作更简化,并且有机试剂消耗少,大大提高了临床适用性和工作效率;选用0.1%甲酸水和0.1%甲酸乙腈为流动相,提高灵敏度,出峰位置稳定,且缩短了单针检测时间;该方法使用同位素标记的内标,方法的耐用性大大提高;此外为了减小该分析方法的基质效应,将蛋白沉淀离心后的上清进行1:1稀释(稀释液:10 %乙腈水)。经方法学验证该方法符合生物样本分析要求,实际样本再分析的通过率为100%,将该方法成功应用于临床血药浓度检测,结果可靠。本试验为阿普斯特相关制剂的生物等效性研究提供了数据参考,适用于阿普斯特的血药浓度检测及其药代动力学研究,为阿普斯特制剂一致性评价提供依据。The method for determining the concentration of apremilast in human blood plasma established by the invention has the advantages of high sensitivity, accuracy and reliability, good selectivity, small matrix effect, quickness and convenience, and the like. This method uses acetonitrile to carry out protein precipitation on plasma samples, and the internal standard working solution is directly prepared into the precipitant, which reduces the operation steps, makes the operation more simplified, and consumes less organic reagents, which greatly improves the clinical applicability and work efficiency; the selection of 0.1 % formic acid water and 0.1% formic acid acetonitrile as the mobile phase, which improves the sensitivity, stabilizes the peak position, and shortens the detection time of a single needle; the method uses an isotope-labeled internal standard, and the durability of the method is greatly improved; in addition, in order to reduce the analysis The matrix effect of the method, the supernatant after centrifugation of the protein pellet was diluted 1:1 (diluent: 10% acetonitrile water). The method was verified by methodology to meet the requirements of biological sample analysis, and the pass rate of actual sample reanalysis was 100%. This method was successfully applied to clinical blood drug concentration detection, and the results were reliable. This test provides a data reference for the bioequivalence study of related preparations of Apremilast, and is suitable for the blood concentration detection and pharmacokinetic research of Apremilast, and provides a basis for the consistency evaluation of Apremilast preparations .
附图说明Description of drawings
图1为加入内标后的含600 ng/mL(定量限)阿普斯特的血浆中阿普斯特的色谱图;Fig. 1 is the chromatogram of apremilast in plasma containing 600 ng/mL (limit of quantification) apremilast after adding internal standard;
图2为加入内标后的含600 ng/mL(定量限)阿普斯特的血浆中内标物质的色谱图;Fig. 2 is the chromatogram of internal standard substance in the plasma containing 600 ng/mL (limit of quantification) apremilast after adding internal standard;
图3为空白血浆中阿普斯特的色谱图;Fig. 3 is the chromatogram of apremilast in blank plasma;
图4为空白血浆中内标物质的色谱图。Figure 4 is a chromatogram of the internal standard substance in blank plasma.
具体实施方式Detailed ways
在接下来的描述中进一步阐述了本发明的具体细节用于充分理解本发明。本发明中的说明书所使用的术语只是为了用于说明本发明的优点和特点,不是旨在于限制本发明。Specific details of the invention are further set forth in the ensuing description for a full understanding of the invention. The terms used in the description of the present invention are only used to describe the advantages and features of the present invention, and are not intended to limit the present invention.
除非另行定义,本发明中所使用的所有专业与科学术语属于本发明的技术领域的技术人员所理解的含义相同。如无特殊说明,本发明所使用的药品或试剂均按照产品说明书使用或采用所属领域的常规使用方法。现根据说明书附图和具体实施方式对本发明的技术方案作进一步说明。Unless otherwise defined, all professional and scientific terms used in the present invention have the same meaning as understood by those skilled in the technical field of the present invention. Unless otherwise specified, the medicines or reagents used in the present invention are used in accordance with product instructions or conventional methods in the field. The technical solution of the present invention will now be further described according to the accompanying drawings and specific embodiments of the specification.
实施例1Example 1
1 .仪器和试药1. Instruments and reagents
1)分析仪器1) Analytical instrument
AB SCIEX Tripe Quad 5500液相色谱-质谱联用系统(美国AB SCIEX公司);3K15台式高速冷冻离心机(德国Sigma公司);Allegra X-15R台式高速冷冻离心机(96孔板,美国贝克曼库尔特公司);MDF-U5412N医用低温箱(日本松下公司);Milli-Q Advantage A10超纯水器(美国Millipore公司);DG-2500R多管漩涡混合仪(上海巴玖实业有限公司)。数据处理软件:Analyst 1.6.3(美国AB SCIEX公司);Watson LIMS 7.5 SPS1(美国赛默飞世尔公司)。AB SCIEX Tripe Quad 5500 liquid chromatography-mass spectrometry system (AB SCIEX, USA); 3K15 desktop high-speed refrigerated centrifuge (Germany Sigma); Allegra X-15R desktop high-speed refrigerated centrifuge (96-well plate, Beckman Library, USA) Walter Company); MDF-U5412N medical low temperature box (Panasonic Company of Japan); Milli-Q Advantage A10 ultrapure water device (Millipore Company of the United States); DG-2500R multi-tube vortex mixer (Shanghai Bajiu Industrial Co., Ltd.). Data processing software: Analyst 1.6.3 (AB SCIEX, USA); Watson LIMS 7.5 SPS1 (Thermo Fisher, USA).
2)试剂2) Reagent
阿普斯特对照品(纯度99.7%,北京曼哈格生物科技有限公司);阿普斯特-d5对照品(化学纯度98.7%,同位素内标纯度99.5 %,北京曼哈格生物科技有限公司);甲醇,乙腈(色谱纯,德国默克公司);甲酸(色谱纯,美国ACS恩科化学);N,N-二甲基甲酰胺(色谱纯,赛默飞世尔科技(中国)科技有限公司)。受试制剂:阿普斯特片(规格:30 mg,批号:02007001);参比制剂:阿普斯特片(规格:30 mg,批号:H02366A,Cegene InternationaSar生产)。Apremilast reference substance (purity 99.7%, Beijing Manhag Biotechnology Co., Ltd.); Apremilast-d5 reference substance (chemical purity 98.7%, isotope internal standard purity 99.5%, Beijing Manhage Biotechnology Co., Ltd. ); methanol, acetonitrile (chromatographically pure, Merck, Germany); formic acid (chromatographically pure, American ACS Enke Chemicals); N, N-dimethylformamide (chromatographically pure, Thermo Fisher Scientific (China) Technology Co., Ltd. Ltd). Test preparation: Apremilast tablet (specification: 30 mg, batch number: 02007001); reference preparation: apremilast tablet (specification: 30 mg, batch number: H02366A, produced by Cegene International Sar).
2.实验方法与结果2. Experimental methods and results
1)溶液配制1) Solution preparation
标准品溶液配制:精密称量1 mg阿普斯特标准品,用N,N-二甲基甲酰胺(DMF)溶解得到1 mg/mL的阿普斯特母液。精密量取1 mL阿普斯特母液适量置10 mL容量瓶中,加50%乙腈水溶液至刻度,使质量浓度为100 µg/mL阿普斯特工作溶液。用50%乙腈水溶液将阿普斯特工作溶液稀释成40、80、200、600,2000,4000,9600,和12000 ng/mL的标准曲线工作液。Preparation of standard solution: Accurately weigh 1 mg of apremilast standard substance and dissolve it in N,N-dimethylformamide (DMF) to obtain 1 mg/mL apremilast mother solution. Precisely measure 1 mL of Apremilast mother liquor and place it in a 10 mL volumetric flask, add 50% acetonitrile aqueous solution to the mark, so that the mass concentration is 100 μg/mL Apremilast working solution. Dilute the apremilast working solution with 50% acetonitrile aqueous solution to 40, 80, 200, 600, 2000, 4000, 9600, and 12000 ng/mL standard curve working solution.
内标工作液配制:精密称量约1 mg阿普斯特-d5标准品,用一定量的N,N-二甲基甲酰胺(DMF)溶解,配成1 mg/mL的内标母液。用乙腈溶液进一步将内标母液稀释为20.0 ng/mL的内标工作液。Preparation of internal standard working solution: Accurately weigh about 1 mg of apremilast-d5 standard substance, dissolve it in a certain amount of N,N-dimethylformamide (DMF), and prepare a 1 mg/mL internal standard mother solution. The internal standard mother solution was further diluted to 20.0 ng/mL internal standard working solution with acetonitrile solution.
2)含阿普斯特的血浆样品预处理2) Pretreatment of plasma samples containing apremilast
取2 mL深孔96孔板,每孔加100 µL含阿普斯特的血浆样品,再加质量浓度为20.0ng/mL内标工作溶液300 µL,涡旋混合5 min,置4℃台式高速冷冻离心机(96孔板)中离心10min(4750 r/min),每孔取100 µL上清液,转移至另一2 mL深孔96孔板,向新的96孔板中加10%乙腈水溶液100 µL稀释,涡旋混合5 min,涡旋混合后将混合液加到插有内插管的进样瓶中,启动液质联用仪检测程序对阿普斯特进行检测。Take a 2 mL deep-well 96-well plate, add 100 µL of plasma samples containing apremilast to each well, and add 300 µL of internal standard working solution with a mass concentration of 20.0 ng/mL, vortex and mix for 5 min, and place at 4 °C on a bench-top high-speed Centrifuge in a refrigerated centrifuge (96-well plate) for 10min (4750 r/min), take 100 µL supernatant from each well, transfer to another 2 mL deep-well 96-well plate, add 10% acetonitrile to the new 96-well plate Dilute with 100 µL of aqueous solution, vortex mix for 5 min, add the mixed solution into the injection bottle with the inner insert tube after vortex mixing, and start the liquid chromatography-mass spectrometer detection program to detect Apremilast.
3)高效液相色谱条件3) High performance liquid chromatography conditions
液质联用检测的高效液相色谱条件包括:色谱柱为Phenomenex Kinetex C18(2.1×50 mm,2.6 µm)色谱柱,采用梯度洗脱,流动相A为含0.1%甲酸的水溶液,流动相B为0.1%甲酸乙腈溶液,洗脱程序为0~0.5 min,流动相A的体积百分含量保持63%,流动相B的体积含量保持37%;0.5~1.3 min,流动相A的体积百分含量从63%下降到0.0%,流动相B的体积百分含量从37%上升到100%;1.3~2.4 min,流动相A的体积百分含量保持0.0%,流动相B的体积含量保持100%;2.4~2.41 min,流动相A的体积百分含量从0.0%上升到63%,流动相B的体积百分含量从100%下降到37%;2.41~3.0 min,流动相A的体积百分含量保持63%,流动相B的体积含量保持37%,流速为0.45 mL/min,柱温为40℃,进样量为5 µL;The HPLC conditions for LC-MS detection include: the chromatographic column is Phenomenex Kinetex C18 (2.1×50 mm, 2.6 μm) chromatographic column, gradient elution is used, mobile phase A is aqueous solution containing 0.1% formic acid, mobile phase B It is 0.1% formic acid acetonitrile solution, the elution program is 0~0.5 min, the volume percentage content of mobile phase A is kept at 63%, and the volume content of mobile phase B is kept at 37%; 0.5~1.3 min, the volume percentage content of mobile phase A is content decreased from 63% to 0.0%, and the volume percentage of mobile phase B increased from 37% to 100%; 1.3~2.4 min, the volume percentage of mobile phase A remained at 0.0%, and the volume content of mobile phase B remained at 100%. %; 2.4~2.41 min, the volume percentage of mobile phase A increased from 0.0% to 63%, and the volume percentage of mobile phase B decreased from 100% to 37%; 2.41~3.0 min, the volume percentage of mobile phase A The component content was kept at 63%, the volume content of mobile phase B was kept at 37%, the flow rate was 0.45 mL/min, the column temperature was 40°C, and the injection volume was 5 µL;
表1液相梯度洗脱程序Table 1 Liquid phase gradient elution program
4)三重四级杆质谱检测条件4) Triple quadrupole mass spectrometry detection conditions
液质联用检测的质谱条件包括:使用三重四级杆质谱仪,离子源为电喷雾离子源,采用负离子模式,采用多反应监测扫描模式,离子化电压:5500 V;入口电压(EP):10 V;碰撞室出口电压(CXP):13 V;喷雾气(Gas1):50;辅助加热气(Gas2):55;气帘气:35,所述阿普斯特的离子对为461.2→257.1(定量离子对),碰撞能量(CE):14 V,去簇电压(DP):190 V;所述阿普斯特-d5的离子对为466.1→262.3,CE:14 V,DP:190 V。The mass spectrometry conditions for liquid chromatography-mass spectrometry detection include: using a triple quadrupole mass spectrometer, the ion source is an electrospray ion source, using negative ion mode, using multiple reaction monitoring scanning mode, ionization voltage: 5500 V; entrance voltage (EP): 10 V; Collision cell outlet voltage (CXP): 13 V; Spray gas (Gas1): 50; Auxiliary heating gas (Gas2): 55; Curtain gas: 35. Quantitative ion pair), collision energy (CE): 14 V, declustering potential (DP): 190 V; the ion pair of Apremilast-d5 is 466.1→262.3, CE: 14 V, DP: 190 V.
5)标准曲线的建立5) Establishment of standard curve
取空白血浆285 µL,加入不同浓度的标准曲线工作液15 µL,得到2.0,4.0,10.0,30.0,100.0,200.0,480.0和600.0 ng/mL的标准曲线样品,取所述标准曲线样品100 µL,加400 µL阿普斯特-d5的乙腈溶液(内标工作溶液)进行沉淀反应,涡旋混合5 min,置4℃台式高速冷冻离心机(96孔板)中,4750 r/min离心10 min,每孔取上清100 µL,转移至另一96孔板,每孔加10%乙腈水溶液100 µL稀释,涡旋混合5 min后进行液质联用检测,使用内标法对所得数据进行处理,以阿普斯特的浓度为横坐标,阿普斯特和阿普斯特-d5的峰面积比值为纵坐标,以1/x2为权重系数,利用加权最小二乘法进行线性回归,求得标准曲线为y=0.0147982*x+-0.00436850(n=9),线性范围是2.00~60000 ng/mL,相关系数r2为0.9977,定量下线为2.00 ng/mL。Take 285 µL of blank plasma, add 15 µL of standard curve working solution of different concentrations to obtain standard curve samples of 2.0, 4.0, 10.0, 30.0, 100.0, 200.0, 480.0 and 600.0 ng/mL, take 100 µL of the standard curve sample, Add 400 µL of acetonitrile solution of Apremilast-d5 (internal standard working solution) to carry out precipitation reaction, vortex and mix for 5 min, put in a desktop high-speed refrigerated centrifuge (96-well plate) at 4°C, and centrifuge at 4750 r/min for 10 min , take 100 µL of supernatant from each well, transfer to another 96-well plate, add 100 µL of 10% acetonitrile aqueous solution to each well to dilute, vortex and mix for 5 min, then perform liquid chromatography-mass detection, and use the internal standard method to process the obtained data , with the concentration of Apremilast as the abscissa, the ratio of the peak areas of Apremilast and Apremilast-d5 as the ordinate, and 1/x 2 as the weight coefficient, use the weighted least squares method to carry out linear regression, find The obtained standard curve was y =0.0147982*x+-0.00436850 (n=9), the linear range was 2.00-60000 ng/mL, the correlation coefficient r2 was 0.9977, and the lower limit of quantification was 2.00 ng/mL.
6)残留6) residual
通过在注射高浓度样品后注射空白基质样品(空白血浆)来评价残留,图1为加入内标后的含600 ng/mL(定量限)阿普斯特的血浆中阿普斯特的色谱图,分析物(阿普斯特)的保留时间为1.17 min;图2为加入内标后的含600 ng/mL(定量限)阿普斯特的血浆中内标物质的色谱图,内标(阿普斯特-d5)的保留时间为1.15 min,均得到较好的响应值与峰形。The residue was evaluated by injecting a blank matrix sample (blank plasma) after injecting a high-concentration sample. Figure 1 is the chromatogram of apremilast in plasma containing 600 ng/mL (quantitative limit) apremilast after adding an internal standard , the retention time of the analyte (Apremilast) was 1.17 min; Figure 2 is the chromatogram of the internal standard substance in plasma containing 600 ng/mL (limit of quantification) Apremilast after adding the internal standard, the internal standard ( Apremilast-d5) had a retention time of 1.15 min, and both obtained good response values and peak shapes.
图3为空白血浆中阿普斯特的色谱图;图4为空白血浆中内标物质的色谱图。将图1和图2与图3和4进行比较,发现空白血浆样品的色谱图在阿普斯特与阿普斯特-d5的色谱峰附近中没有干扰峰出现,说明该方法的残留小,对分析物(阿普斯特)及内标(阿普斯特-d5)的定量均无影响。Fig. 3 is the chromatogram of apremilast in blank plasma; Fig. 4 is the chromatogram of internal standard substance in blank plasma. Comparing Fig. 1 and Fig. 2 with Fig. 3 and 4, it is found that the chromatogram of the blank plasma sample has no interference peaks near the chromatographic peaks of Apremilast and Apremilast-d5, indicating that the residue of this method is small, There was no effect on the quantification of the analyte (Apremilast) and the internal standard (Apremilast-d5).
7)精密度和准确度考察7) Inspection of precision and accuracy
按标准曲线样品的配制方法,分别配制含2.0、6.0、50.0、450 ng/mL阿普斯特的质控样品,每个浓度独立配制6个样本,连续检测3天。根据当天配制的标准曲线,计算质控样品的浓度,进而计算批内、批间精密度和准确度,结果如表2和表3所示。从表2的数据可以看出,本发明公开的方法的批内、批间准确度和精密度均在±15%范围以内,说明该方法准确且可重复,适合推广。According to the standard curve sample preparation method, quality control samples containing 2.0, 6.0, 50.0, and 450 ng/mL apremilast were prepared respectively, and 6 samples were independently prepared for each concentration, and tested continuously for 3 days. According to the standard curve prepared on the same day, the concentration of the quality control sample was calculated, and then the intra-assay and inter-assay precision and accuracy were calculated. The results are shown in Table 2 and Table 3. It can be seen from the data in Table 2 that the intra-assay and inter-assay accuracy and precision of the method disclosed in the present invention are within the range of ±15%, indicating that the method is accurate and repeatable, and is suitable for promotion.
表2准确度与精密度实验结果Table 2 Accuracy and precision experimental results
8)选择性8) Optional
取6个受试者的空白血浆制备空白基质样品与定量下限样品,空白基质样品除用300 µL乙腈溶液代替内标工作溶液外,其余步骤按“含阿普斯特的血浆样品预处理”项处理样品,定量下限样品按“含阿普斯特的血浆样品预处理”项处理样品。结果表明本研究6个受试者的空白基质干扰组分在阿普斯特与阿普斯特-d5保留时间处的响应均为0,表明该方法具有良好的选择性,能区分阿普斯特与阿普斯特-d5与基质中的内源性组分。Take the blank plasma of 6 subjects to prepare the blank matrix sample and the lower limit of quantification sample. Except for the blank matrix sample, 300 μL acetonitrile solution was used to replace the internal standard working solution, and the rest of the steps were as in the "Plasma Sample Pretreatment Containing Apremilast" item. Samples were processed, samples with the lower limit of quantitation were processed according to the item "Plasma Sample Pretreatment Containing Apremilast". The results showed that the responses of the blank matrix interference components of the 6 subjects in this study were all 0 at the retention time of Apremilast and Apremilast-d5, indicating that the method had good selectivity and could distinguish Apremilast Tetra and apremilast-d5 and endogenous components in the stroma.
9)提取回收率和基质效应考察9) Investigation of extraction recovery and matrix effect
回收率:将质控样品按“含阿普斯特的血浆样品预处理”项处理,得峰面积A;取100µL空白血浆,加入300 µL乙腈沉淀剂,混匀离心后取上清液100到新的2 mL深孔96孔板,再向新的96孔板中加100 µL 混合对照品,涡旋混合5 min,混匀后进样检测,得峰面积B。峰面积A与B的比值为回收率,结果见表3。Recovery rate: The quality control sample was processed according to the item "Plasma Sample Pretreatment Containing Apremilast" to obtain the peak area A; take 100 µL of blank plasma, add 300 µL of acetonitrile precipitant, mix and centrifuge, and take the supernatant 100 to A new 2 mL deep-well 96-well plate was added, and 100 µL of the mixed reference substance was added to the new 96-well plate, vortexed for 5 min, mixed evenly and injected for detection, and the peak area B was obtained. The ratio of the peak area A to B is the recovery rate, and the results are shown in Table 3.
表3提取回收率实验结果Table 3 extraction recovery experimental results
基质效应:分别取6个受试者的空白基质、1个高血脂空白基质及1个溶血各100 µL,加入300 µL乙腈沉淀剂,混匀离心后取上清液100 µL到新的2 mL深孔96孔板,再向新的96孔板中加100 µL混合对照品,涡旋混合5 min,混匀后进样检测,得分析物峰面积C,内标峰面积D。取100 µL纯水,加入300 µL乙腈沉淀剂,混匀离心后取上清液100 µL到新的2 mL深孔96孔板,再向新的96孔板中加100 µL混合对照品,涡旋混合5 min,混匀后进样检测,得分析物峰面积E,内标峰面积F。基质效应用内标归一化的基质因子的变异系数(CV)来评价。基质因子为基质存在下的峰面积与不含基质的相应峰面积比值,即阿普斯特的基质因子为C/E,阿普斯特-d5的基质因子为D/F。进一步通过阿普斯特的基质因子除以阿普斯特-d5的基质因子,计算经内标归一化的基质因子,再计算内标归一化的基质因子的变异系数,结果见表4。Matrix effect: Take 100 µL each of the blank matrix, 1 hyperlipidemia blank matrix, and 1 hemolysis of 6 subjects, add 300 µL of acetonitrile precipitant, mix well and centrifuge, and transfer 100 µL of the supernatant to a new 2 mL Deep-well 96-well plate, then add 100 μL mixed reference substance to a new 96-well plate, vortex mix for 5 min, mix well and inject sample for detection, get analyte peak area C and internal standard peak area D. Take 100 µL of pure water, add 300 µL of acetonitrile precipitant, mix well and centrifuge, transfer 100 µL of the supernatant to a new 2 mL deep-well 96-well plate, then add 100 µL of the mixed control substance to the new 96-well plate, vortex Rotate and mix for 5 min, inject the sample for detection after mixing, and obtain the peak area E of the analyte and the peak area F of the internal standard. The matrix effect was evaluated by the coefficient of variation (CV) of the matrix factor normalized by the internal standard. The matrix factor is the ratio of the peak area in the presence of matrix to the corresponding peak area without matrix, that is, the matrix factor of Apremilast is C/E, and the matrix factor of Apremilast-d5 is D/F. Further, by dividing the matrix factor of Apremilast by the matrix factor of Apremilast-d5, the matrix factor normalized by the internal standard was calculated, and then the coefficient of variation of the matrix factor normalized by the internal standard was calculated. The results are shown in Table 4 .
表4 6批不同来源血浆、高脂及溶血的基质效应Table 4 Matrix effect of 6 batches of different sources of plasma, high fat and hemolysis
从以上结果可以看出,本发明公开的基于沉淀法和液质联用技术的血浆中阿普斯特定量检测法能够快速、稳定地检测生物样本中阿普斯特的浓度。It can be seen from the above results that the quantitative detection method for apremilast in plasma based on the precipitation method and liquid chromatography-mass spectrometry technology disclosed by the present invention can quickly and stably detect the concentration of apremilast in biological samples.
以上所述仅是本发明的优选实施方式,并非对本发明作任何形式上的限制。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。The above descriptions are only preferred embodiments of the present invention, and do not limit the present invention in any form. It should be pointed out that those skilled in the art can make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.
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Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017084597A1 (en) * | 2015-11-19 | 2017-05-26 | 常州爱诺新睿医药技术有限公司 | Amorphous form of apremilast, preparation method therefor, and application thereof |
EP3623816A1 (en) * | 2018-09-12 | 2020-03-18 | GLX Analytix ApS | Glx-derived molecule detection |
CN111198235A (en) * | 2020-01-13 | 2020-05-26 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | A kind of detection method of isoflavone content in plasma |
CN111665314A (en) * | 2020-07-10 | 2020-09-15 | 江苏知原药业有限公司 | Method for detecting impurity 1 and impurity 2 in Apremilast tablet |
CN112305107A (en) * | 2020-10-23 | 2021-02-02 | 杭州朱养心药业有限公司 | Apremilast composition of phosphodiesterase-4 inhibitor and quality detection method |
WO2021262956A1 (en) * | 2020-06-24 | 2021-12-30 | Roivant Sciences Gmbh | Topical gel formulations and their use in treating skin conditions |
CN113960208A (en) * | 2021-10-28 | 2022-01-21 | 济南良福精合医药科技有限公司 | A kind of method for measuring active ingredient content in apremilast-containing preparation |
CN114577957A (en) * | 2021-12-21 | 2022-06-03 | 益诺思生物技术南通有限公司 | A kind of detection method of voriconazole drug in iris |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11357775B2 (en) * | 2019-04-30 | 2022-06-14 | Celgene Corporation | Combination therapies comprising apremilast and Tyk2 inhibitors |
-
2022
- 2022-11-28 CN CN202211497301.0A patent/CN115541778B/en active Active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017084597A1 (en) * | 2015-11-19 | 2017-05-26 | 常州爱诺新睿医药技术有限公司 | Amorphous form of apremilast, preparation method therefor, and application thereof |
EP3623816A1 (en) * | 2018-09-12 | 2020-03-18 | GLX Analytix ApS | Glx-derived molecule detection |
CN111198235A (en) * | 2020-01-13 | 2020-05-26 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | A kind of detection method of isoflavone content in plasma |
WO2021262956A1 (en) * | 2020-06-24 | 2021-12-30 | Roivant Sciences Gmbh | Topical gel formulations and their use in treating skin conditions |
CN111665314A (en) * | 2020-07-10 | 2020-09-15 | 江苏知原药业有限公司 | Method for detecting impurity 1 and impurity 2 in Apremilast tablet |
CN112305107A (en) * | 2020-10-23 | 2021-02-02 | 杭州朱养心药业有限公司 | Apremilast composition of phosphodiesterase-4 inhibitor and quality detection method |
CN113960208A (en) * | 2021-10-28 | 2022-01-21 | 济南良福精合医药科技有限公司 | A kind of method for measuring active ingredient content in apremilast-containing preparation |
CN114577957A (en) * | 2021-12-21 | 2022-06-03 | 益诺思生物技术南通有限公司 | A kind of detection method of voriconazole drug in iris |
Non-Patent Citations (1)
Title |
---|
LC-MS/MS Determination and Pharmacokinetics Study of Apremilast after Oral Administration in Rabbits;Tammisetty Mohan Rao et al.;《Indian Journal of Pharmaceutical Education and Research》;20200630;第54卷(第2期);第440-447页 * |
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