CN105806973B - Uplc-ms/ms法测定人血浆中莎巴比星及其代谢产物m3的血药浓度 - Google Patents
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Abstract
本文涉及UPLC‑MS/MS法测定人血浆中莎巴比星及其代谢产物M3的血药浓度。本文提供了通过超高效液相色谱串联质谱法(UPLC‑MS/MS)测定人血浆中莎巴比星和C13醇代谢产物M3的浓度的方法,其中所述方法采用甲酸水‑乙腈作为流动相进行。本文提供的莎巴比星和代谢物M3检测方法灵敏度高、重复性好,可以满足莎巴比星治疗恶性实体瘤的临床药代动力学生物样品定量分析的要求。
Description
技术领域
本发明涉及检测人血浆中药物的浓度的方法。具体而言,本发明涉及通过超高效液相色谱串联质谱法(UPLC-MS/MS)测定人血浆中莎巴比星和C13醇代谢产物M3的浓度的方法。
背景技术
引言
莎巴比星属于蒽环类药物,为意大利美纳里尼制药公司通过延长和修饰碳水化合物而合成的新型药物,具有更强的药效以及更低的心脏毒性[11,12]。动物实验表明,莎巴比星对不同组织来源肿瘤的抑制作用比它的同类药物多柔比星更强[13]。而且欧洲的I、II期临床试验已初步验证莎巴比星在人体的安全性和耐受性,证明莎巴比星在卵巢癌和肺癌中具有较好的疗效[11]。本研究旨在建立灵敏、快捷的超高效液相色谱质谱联用(UPLC-MS/MS)方法检测人血浆的莎巴比星和代谢物M3的血药浓度,为莎巴比星治疗难治或复发的SCLC的I期临床药代动力学研究提供支持。
检测血液或其他生物样本中的药物浓度是药动学、药效学研究和临床治疗的基础。用于检测血浆药物浓度的方法应当具备高灵敏度、能够快速获得结果和能够使用少量样品进行。然而,由于血液中存在包括磷脂、蛋白等多种组份,检测易受基质效应等各种因素的影响。如何最大可能减少基质效应,尽可能获得血液中药物的准确浓度,是本领域必须进行深入研究的课题。
HPLC法是目前检测血药浓度常用的方法,应用范围广泛。但是,由于使用色谱柱进行分离耗费时间较长、灵敏度较低,并不适用于高通量的生物样品分析。
超高效液相色谱质谱联用(Ultra Performance Liquid Chromatography,UPLC-MS/MS)法提高了分析通量,节省了样品和溶剂分析时间。然而,UPLC-MS/MS对流动相,色谱柱,样品前处理,质谱接口,数据采集系统都提出了特别的要求。尽管对转换方法进行了一些研究,传统的高效液相色谱HPLC的条件不能直接应用于UPLC-MS/MS,需要对包括样品前处理、内标、流动相、色谱柱选择、质谱条件设置等各项条件进行大量试验,才能实现UPLC-MS/MS快速检测分析。例如,周新等人的研究(HPLC与UPLC色谱条件转换方法研究,分析试验室,第27卷第4期,2008年4月)表明直接将HPLC条件应用于UPLC,不能达到对待测物质分离分析的目的;而且质谱检测与色谱检测不同,对于流动相组成、流速等多种方面需要额外的注意,因此对实验条件包括例如流动相选择等进行具体深入研究才能获得分离状况良好的UPLC-MS/MS方法。
目前对于心脏毒性的机制研究显示,C13醇代谢产物可通过影响心肌细胞Ca2+交换和能力代谢而损伤心肌细胞。并且相对于原形药,C13醇代谢产物在心肌细胞内蓄积时间更长,其蓄积浓度与心脏毒性具有相关性[15]。因此对于莎巴比星的C13醇代谢产物M3的药代动力学研究对预测莎巴比星的心脏毒性具有重要价值。建立莎巴比星和代谢物M3血药浓度的检测方法对于莎巴比星的临床研究具有重要意义。目前尚未见有用于测定人血浆中莎巴比星和C13醇代谢产物M3的浓度的方法的报道。已经报道方法不能同时检测C13醇代谢产物M3,灵敏度较低,不适用于临床药代动力学研究的大批量的样本检测。
本领域急需能够用于同时检测人血浆中莎巴比星和M3的浓度的快速、灵敏的方法。
发明内容
发明人已经建立了快速、灵敏的超高效液相色谱串联质谱(ultra highperformance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)技术检测人血浆中莎巴比星和C13醇代谢产物M3的浓度。本发明的方法具有的优点包括例如样本处理简单、快捷、灵敏度高等。鉴于C13醇代谢产物相对于原形药在心肌细胞内蓄积时间更长,其蓄积浓度与心脏毒性具有相关性,对于莎巴比星的C13醇代谢产物M3的药代动力学研究对预测莎巴比星的心脏毒性具有重要价值。本发明实现了对人血浆中莎巴比星和C13醇代谢产物M3的浓度的同时检测,具有重要意义。
在一些实施方案中,本发明提供通过超高效液相色谱串联质谱法(UPLC-MS/MS)测定人血浆中莎巴比星和C13醇代谢产物M3的浓度的方法,其中所述方法采用甲酸水-乙腈作为流动相进行。发明人已经发现采用甲酸水-乙腈作为流动相进行分析时,洗脱能够快速、充分分离待测物质,获得良好的峰形,基线噪音低。
在一些实施方案中,本发明的方法采用的甲酸水-乙腈流动相的体积比选自以下范围:甲酸水:乙腈(90/10,v/v)-甲酸水:乙腈(10/90,v/v),甲酸水:乙腈(80/20,v/v)-甲酸水:乙腈(20/80,v/v),甲酸水:乙腈(70/30,v/v)-甲酸水:乙腈(30/70,v/v),甲酸水:乙腈(60/40,v/v)-甲酸水:乙腈(40/60,v/v)。
在一些实施方案中,本发明的方法中采用的流动相为0.1%甲酸水-乙腈。在一些实施方案中,所述流动相采用上述体积比进行。在一些实施方案中,本发明的方法中采用梯度洗脱进行。在一些实施方案中,本发明的方法采用下述梯度洗脱进行A:0.1%甲酸水;B:乙腈,洗脱梯度为:0~0.5min:10%B;0.51~2.8min:28%B;2.81~3.8min:90%B,3.81~4.5min:10%B。在一些实施方案中,洗脱流速设为0.3ml·min-1。为了改善色谱分离选择性,可以考虑调节流动相的极性,向流动相中加入改性剂等。已经观察到采用所述流动相进行洗脱能够充分分离待测物质,获得良好的峰形和较快的分析时间。
在一些实施方案中,本发明的方法采用ACQUITY UPLC BEH Shield RP18(100mm×2.1mm,1.7μm)作为分析柱进行。在色谱法中,色谱柱的选择十分重要,对色谱柱的要求是柱效高、选择性好,分析速度快等。在本发明中,发明人发现ACQUITY UPLC BEH Shield RP18(100mm×2.1mm,1.7μm)能够实现莎巴比星和C13醇代谢产物M3良好分离和峰型,但是洗脱时间适当。因此,已经观察到采用ACQUITY UPLC BEH Shield RP18(100mm×2.1mm,1.7μm)作为分析柱能够充分分离待测物质,获得良好的峰形和较快的分析时间。
在一些实施方案中,本发明的方法可以采用多柔比星为内标进行。在采用内标法时,内标物的选择是一项十分重要的工作。理想的内标物应当能以准确、已知的量加到样品中去,和被分析的样品有基本相同或尽可能一致的物理化学性质(如化学结构、极性、挥发度及在溶剂中的溶解度等)、色谱行为和响应特征;在色谱分析条件下,内标物必须能与样品中各组分充分分离。在一些实施方案中,发明人已经显示采用多柔比星作为内标能够提供线性关系良好、精密度、基质效应、提取回收率以及稳定性符合要求的检测方法。
血浆样品的前处理过程是UPLC-MS/MS中获得高灵敏度的关键。在本发明中,可采用蛋白沉淀法、固相萃取法和液液萃取法对血浆样品进行处理。然而,本发明的方法优选采用液液萃取法进行血浆样本的预处理。本发明提供的数据表明采用液液萃取法简便、灵敏,液液萃取法完全满足试验中对测定的要求。在本发明中,可以通过例如氯仿异丙醇进行萃取。氯仿和异丙醇的体积比没有特别的限定。在一些实施方案中,氯仿:异丙醇的体积比可以选自下述范围:氯仿:异丙醇(90/10,v/v)-氯仿:异丙醇(10/90,v/v),氯仿:异丙醇(80/20,v/v)-氯仿:异丙醇(20/80,v/v),氯仿:异丙醇(70/30,v/v)-氯仿:异丙醇(30/70,v/v),氯仿:异丙醇(60/40,v/v)-氯仿:异丙醇(40/60,v/v)。在一些实施方案中,可以使用氯仿:异丙醇(50/50,v/v)。在一些实施方案中,在加入氯仿异丙醇之前,可以向血浆样品中加入正己烷进行处理。本发明的方法可以采用较低的样品体积进行。在本发明的条件下,令人吃惊地发现较低的样品完全能够满足分析需要。本发明的方法已经证明在本发明的条件下显著低于文献报道的血浆样本体积如30μl-200μl,例如35μl-180μl,40μl-160μl,50μl-150μl,60μl-120μl的体积即能够满足分析需要,因此特别适合于临床试验中较少的血浆样本体积分析的目的。
优选地,在本发明的条件下可以采用液液萃取法进行血浆样品处理。已经发现最初血浆样品用液液萃取法处理后,进一步用甲醇水复溶后进样。在一些实施方案中,本发明的方法能够获得较高的回收率。发明人发现液液萃取法处理后加入甲醇水复溶,可以获得更好的峰形,并减小残留,尤其能够获得提高的灵敏度。在一些实施方案中,本发明的方法检测血浆中的莎巴比星和M3的LLOQ分别为2ng·mL-1和0.5ng·mL-1,且具有良好的重复性。鉴于莎巴比星给药后莎巴比星和M3的浓度较低,足够高的灵敏度对于检测莎巴比星和M3是必需的。
在一些实施方案中,本发明的方法具有提高的灵敏度。在一些实施方案中,本发明的方法检测血浆中的莎巴比星和M3的LLOQ分别为2ng·mL-1和0.5ng·mL-1。在一些实施方案中,本发明的方法检测的莎巴比星和M3在2~400ng·ml-1和0.5~100ng·ml-1范围内线性较好。
在一些实施方案中,本发明的方法中所述流动相采用0.1-0.8ml/min的流速进行,优选采用0.2-0.50ml/min的流速进行。
在一些实施方案中,本发明的方法可以采用梯度洗脱进行。
在一些实施方案中,血浆样品前处理采用正己烷进行处理,通过例如氯仿异丙醇进行萃取,经甲醇水复溶后用ACQUITY UPLC BEH Shield RP18(100mm×2.1mm,1.7μm)色谱柱进行分析。
本发明的方法的特异性、基质效应、提取回收率以及稳定性均进行了验证,所述方法已经成功应用于临床药代动力学样本的检测。
本研究建立的莎巴比星和代谢物M3检测方法灵敏度高、重复性好,可以满足莎巴比星治疗恶性实体瘤的临床药代动力学生物样品定量分析的要求。
附图说明
图1:莎巴比星(左)和代谢物M3(右)的化学结构式。
图2:二级质谱图A:莎巴比星;B:M3;C:内标(多柔比星)。
图3:空白血浆、含内标的空白血浆和LLOQ样品的特征性色谱图。
图4:莎巴比星和M3药时曲线图。
具体实施方式
为了更加清楚的对本发明进行说明,以下通过具体实施实例对本发明的具体实施方式进行更为详细的说明。但是应该理解,以下所述具体实施实例仅用于本发明进行示例性说明,而非用于本发明进行任何性质限定,其中所用材料、试剂、仪器及操作条件仅为代表性的,其并不限于所列举的情况。所属技术领域的技术人员通过阅读以下说明可以对本发明做出不脱离本发明权利要求所限定的保护范围的改动和改进,这些改动和改进也处于本发明所要求保护的范围内。
1、发明人已经建立超高效液相色谱质谱联用(UPLC-MS/MS)检测人血浆中莎巴比星和代谢物M3浓度的方法。液相条件可以选用ACQUITY UPLC BEH Shield RP18(100mm×2.1mm,1.7μm),流动相为A:0.1%甲酸水;B:乙腈,0.3mL·min-1梯度洗脱。质谱方法采用电喷雾电离,正离子多反应监测模式,监测离子对:莎巴比星m/z 644→130、M3m/z 646→333.2和内标m/z 544→360。按照2015年《中国药典》中《生物样品定量分析方法验证指导原则》进行了方法学的考察。检测1例受试者样品,初步验证方法的适用性。莎巴比星和M3在2~400ng·ml-1和0.5~100ng·ml-1范围内线性较好,r>0.99,莎巴比星、代谢物M3批间和批内精密度范围分别为1.5%~9.1%和2.0%~12.8%,准确度范围分别为-9.6%~1.6%和-4.8%~5.9%;莎巴比星、M3和内标的提取回收率分别为62.4%,71.9%和58.8%,基质效应可接受。血浆样品室温放置3h和6h,反复冻融3次,进样器放置24h均稳定。20mg·m2-1剂量组受试者,莎巴比星峰浓度为738ng·mL-1,M3峰浓度为3.17ng·mL-1。2、材料与方法
2.1、试剂与药品
莎巴比星(批号:20110502纯度99.64%)和M3(批号:150420纯度92%)(化学结构见图1)标准品由合肥合源药业有限公司提供,内标(Internal Standard,IS)盐酸多柔比星购自中国食品药品检定研究院(批号:130509-201302,纯度100%),甲醇(美国Fhisher公司,HPLC级),乙腈(美国Fhisher公司,HPLC级),异丙醇(美国Fhisher公司,HPLC级),甲酸(美国Fhisher公司,HPLC级),氯仿(分析纯,北京化工厂);实验用水为MILLI-Q Direct 8超纯水处理系统(美国密理博公司)所制纯化水。
2.2液质条件
液相条件:ACQUITY UPLC BEH Shield RP18(100mm×2.1mm,1.7μm)色谱柱,柱温设为40℃,流动相为A:0.1%甲酸水;B:乙腈,洗脱梯度为:0~0.5min:10%B;0.51~2.8min:28%B;2.81~3.8min:90%B,3.81~4.5min:10%B流速设为0.3ml·min-1,进样量为10μL。
质谱条件:选用电喷雾电离,正离子、MRM模式。喷雾电压:5500V,离子源温度:500℃,气帘气(CUR GAS)40psi,雾化气(GAS1)为40psi,辅助加热干燥气(GAS2)为40psi。莎巴比星、代谢产物M3和内标多柔比星主要生成[M+H]+准分子离子峰,MRM监测的离子对分别为:莎巴比星m/z 644→130,M3m/z 646→333.2,多柔比星m/z 544→360(二级质谱扫描图见图2)。莎巴比星的碰撞能(CE)为32,DP、EP及CXP值分别为135V、10V、13V。M3的CE为40,DP、EP及CXP值分别为135V、10V、13V。内标多柔比星CE为35,DP、EP及CXP值分别为90V、10V、13V。
2.3标准品溶液的配制
分别称量约10mg盐酸莎巴比星、M3和盐酸多柔比星标准品于50mL离心管中,根据标准品纯度和分子式计算出称得的标准品中莎巴比星、M3和多柔比星的含量,加入相应体积的甲醇,配置成浓度为1mg·mL-1的标准品储备液。以甲醇稀释获得500ng·mL-1的内标工作液。
以甲醇将莎巴比星储备液和M3储备液分别稀释成400μg/ml和100μg/ml,等体积混合后进一步稀释为4000/1000ng/ml,2000/500ng/ml,1000/250ng/ml,500/125ng/ml,200/50ng/ml,100/25ng/ml,50/10ng/ml,20/5ng/ml的标准曲线工作液,及3000/750ng/ml,400/100ng/ml,40/10ng/ml的质控工作溶液。
2.4样品的前处理
2.0mL Ep管中加入40μL内标工作液和10μL工作液,常温吹干,加入100μL空白血浆后涡旋混匀20s,再加入1mL正己烷涡旋1min,静置2min后,弃上清,再加入1mL氯仿:异丙醇1∶1的萃取液进行液液萃取,涡旋1min,12000rpm离心10min,吸取900μL下层有机相于新的Ep管内,25℃吹干后加入180μL 50%甲醇水复溶,涡旋30s,12000rpm离心10min,取上清进样检测。
2.5方法学考察内容
根据2015年新版《中国药典》中《生物样品定量分析方法验证指导原则》对检测方法进行方法学验证,以确保检测的准确性、可重复性和稳定性。验证包括以下内容:特异性、标准曲线、精密度和准确度、基质效应、提取回收率、稳定性。
2.6.临床试验样本适用性实验
莎巴比星用于难治或复发的SCLC的Ⅰ期临床研究在中国医学科学院肿瘤医院进行(临床批件号:2013L00980)。试验入选一线治疗失败的广泛期SCLC患者。给药剂量设计采用改良的Fibonacci法递增,起始剂量为20mg·m2-1,剂量递增依次为:20mg·m2-1、40mg·m2-1、60mg·m2-1、80mg·m2-1、100mg·m2-1。在给药前0h,滴注中20、40和60min,给药后30min、1、2、4、8、12、24、48、60、72和96h采取静脉血进行药代动力学研究。静脉血采集后立即3000rpm,4℃离心10min,上层血浆分装后冻存于-80℃冰箱中。通过检测1例20mg·m2-1剂量组受试者血药浓度,初步验证检测方法的适用性。
3.结果与讨论
3.1特异性
以6个个体的空白血浆分别配制空白样品、含内标的空白样品以及低定量下限(Lower Limit Of Quantitation,LLOQ)浓度(莎巴比星2ng·mL-1,M30.5ng·mL-1)的样品。三种样品经前处理后进样检测获得谱图(特征性谱图如图3)。如图所示,莎巴比星、M3和内标的保留时间分别为:2.08,1.82和1.76min。在保留时间附近均未见有干扰峰,LLOQ浓度时信噪比均大于10。
3.2标准曲线
10μL标准曲线工作液加40μL内标工作液在2.0mL Ep管中,常温吹干,再加入100μL空白血浆,配置成莎巴比星/M3浓度为:2/0.5、5/1.25、10/2.5、20/5、50/12.5、100/25、200/50、400/100ng·mL-1的标准曲线。考察三个批次的标准曲线,用1/x2加权线性回归计算标准曲线。莎巴比星和M3在2~400ng·mL-1和0.5~100g·mL-1范围线性良好,相关系数r均大于0.99。
3.3精密度和准确度
LLOQ、低、中、高质控浓度样品各6个,经处理后进样检测。通过测得浓度的相对标准偏差(RSD%)和准确度偏倚(Bias%)考察1个检测批内以及多个检测批间的精密度和准确度。莎巴比星和M3,3个浓度的批内和批间RSD%和Bias%均在15%以内,LLOQ浓度均在20%以内,方法定量的精密度和准确度较好,符合指导原则的要求(见表1)。
表格1莎巴比星和M3批内和批间精密度和准确度
3.4基质效应和提取回收率
40μl IS工作液和10μl QC工作液加入到150μl 1∶2甲醇水配置成SET1,进样测得莎巴比星、M3和内标的峰面积分别记为A1、B1和C1;以6个个体血浆经前处理后再加入40μlIS工作液和10μl QC工作液以及150μl 1:2甲醇水配置成SET 2,进样获得莎巴比星、M3和内标的峰面积A2、B2和C2;按照血浆样品处理,即取40μl IS工作液和10μl QC工作液于2.0mL离心管中常温吹干,再加入100μL6个个体血浆,配置成QC样品。随后以1mL正己烷提取后弃上清,再加入氯仿:异丙醇1:1的提取液1mL,涡旋1min,并1200rpm离心10min,随后取下层有机相900μL于新的Ep管中,吹干、复溶,进样测得到峰面积A3、B3和C3。以A、B、C3/A、B、C2·100%计算得到三个化合物的提取回收率。以A、B、C2/A、B、C1·100%计算得到基质效应。不同个体间莎巴比星和M3基质效应的RSD%在各浓度均小于15%,基质效应可接受。莎巴比星、M3和内标的平均提取回收率分别为62.4%,71.9%和58.8%r。(数据见表2)
表格2莎巴比星、M3和内标的基质效应和提取回收率
“*”数据为内标的基质因子的RSD%
3.5稳定性
3.5.1室温稳定性
低、中、高质控样品5份,避光置于室温3、6h,处理后进样检测,以新鲜配制的标准曲线进行定量,考察血浆样品的室温稳定性。结果莎巴比星和M3室温避光放置3h和6h稳定。
3.5.2反复冻融稳定性
低、中、高质控样品各5份置于-80℃冻存24h后取出于室温融化,反复3次,第3次取出融化后处理,进样检测莎巴比星和M3-80℃和室温反复冻融3次稳定。
3.5.3进样器稳定性
低、中、高质控各5份处理后置于样品管理器中24h,温度设为4℃,24h后以新鲜制备的标准曲线定量。莎巴比星和M3在处理后溶液中进样器4℃放置24h稳定。
表格3莎巴比星和M3稳定性数据
3.6临床应用
20mg·m2-1剂量组SCLC受试者莎巴比星血药浓度在给药结束后即刻达峰,峰浓度(Cmax)为738ng/ml,可测到给药后72h;M3在给药后12h达峰,Cmax为3.17ng/ml,至给药后60h仍可测得血药浓度。莎巴比星和M3药时曲线图见图4。
讨论
心脏毒性为蒽环类药物的剂量限制性毒性[14],目前对于心脏毒性的机制研究显示,C13醇代谢产物可通过影响心肌细胞Ca2+交换和能力代谢而损伤心肌细胞。并且相对于原形药,C13醇代谢产物在心肌细胞内蓄积时间更长,其蓄积浓度与心脏毒性具有相关性[15]。因此对于莎巴比星的C13醇代谢产物M3的药代动力学研究对预测莎巴比星的心脏毒性具有重要价值。故建立莎巴比星和代谢物M3血药浓度的检测方法对于莎巴比星的临床研究具有重要意义。
目前对于莎巴比星的检测,用UPLC-MS/MS方法检测人血浆中莎巴比星和C13醇代谢产物M3的方法仍未见有文献报道。本研究开发的方法大幅度缩短了检测时间并减小了生物样品的使用量。目前的方法的莎巴比星和M3的LLOQ分别为2ng·mL-1和0.5ng·mL-1,且具有良好的重复性。
根据预实验的结果,给药剂量为20mg·m2-1时,莎巴比星Cmax为738ng/ml,现有的LLOQ可测到给药后72h的血药浓度。而M3的浓度较低,峰浓度为3.17ng/ml,0.5ng·mL-1的LLOQ并不能满足检测到M3Cmax的1/10。但是由于20mg·m2-1为初始剂量,剂量较低,代谢物M3的浓度可随着后续给药剂量增加而增大。且根据Perez-Blanco JS等的研究,多柔比星给药剂量为50~65mg·m2-1时,其C13醇代谢产物的的血药浓度范围为4.8~104.9ng·mL-1[16]。而相对于多柔比星,莎巴比星代谢产生的C13醇代谢产物更低[17],因此0.5~100ng·mL-1的线性范围可满足I期临床药代动力学定量检测的需要。本研究首次建立的用UPLC-MS/MS检测人血浆中莎巴比星和代谢物M3的方法,灵敏、稳定且重复性好,可较好的应用于莎巴比星治疗恶性实体瘤的I期临床药代动力学研究生物样品的检测。
参考文献
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Claims (3)
1.通过超高效液相色谱串联质谱法UPLC-MS/MS测定人血浆中莎巴比星和C13醇代谢产物M3的浓度的方法,其特征在于,待测人血浆样品通过氯仿异丙醇液液萃取法进行前处理,氯仿:异丙醇的体积比为60/40至40/60,通过液液萃取法进行前处理后,进一步用甲醇水复溶,并且所述方法采用ACQUITY UPLC BEH Shield RP18,100mm×2.1mm,1.7μm分析柱,以甲酸水-乙腈作为流动相采用下述梯度进行洗脱,甲酸水:乙腈体积比为90/10至10/90,A:0.1%甲酸水;B:乙腈,洗脱梯度为:0~0.5min:10%B;0.51~2.8min:28%B;2.81~3.8min:90%B,3.81~4.5min:10%B,所述流动相的流速为0.2-0.50ml/min。
2.如权利要求1所述的方法,其特征在于所述方法采用多柔比星为内标进行。
3.如权利要求1或2所述的方法,其特征在于所述方法检测莎巴比星和M3的LLOQ分别为2ng·mL-1和0.5ng·mL-1。
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