CN111337602A - 一种高效液相色谱串联质谱法测定神经甾体类激素的方法 - Google Patents
一种高效液相色谱串联质谱法测定神经甾体类激素的方法 Download PDFInfo
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Abstract
本发明公开了一种高效液相色谱串联质谱法测定神经甾体类激素的方法,涉及动物体外激素测定方法领域。其方法包括以下步骤:称取神经甾体标准品配制成标准溶液;取待测样品,称重后加入乙腈和MT,涡旋并离心,取上清液,将上清液加入至预先用乙腈清洗过的EMR固相萃取小柱中过滤,得净化后的样品溶液;利用高效液相色谱‑串联质谱仪分别对标准溶液和样品溶液进行检测分析。本发明通过乙腈沉淀蛋白提取后过EMR小柱的方法去除蛋白等干扰成分,解决AP的标准溶液不成线性,响应极低的问题;同时使得测定的各神经甾体具有较好的回收率。
Description
技术领域
本发明涉及动物体外激素测定方法,具体涉及一种高效液相色谱串联质谱法测定神经甾体类激素的方法。
背景技术
神经甾体是指在神经***中合成的甾体和经血脑屏障进入神经***发挥作用的外周甾体及其代谢衍生物。这些神经甾体可以通过结合不同的神经受体(如GABAA,Sigma受体)调控神经功能。神经甾体的行为与中枢神经***正常功能的行使密切相关。研究表明,在不同的生理和病理条件下,神经甾体在各个脑区中的含量会显著变化。神经甾体还会参与焦虑和抑郁的调节过程。由于神经甾体在生物体内含量极低,基质干扰多,使得生物体内内源性神经甾体的测定十分困难,因而开发快速、准确检测生物体内神经甾体含量的方法就显得尤为重要。
传统的神经甾体的检测方法有放射免疫标记法(RIA)和气相色谱质谱联用法(GC-MS),但放射免疫标记法特异性差,且存在安全隐患;气相色谱-质谱联用法虽然灵敏度高,但样品前处理步骤复杂,不能实现高通量分析。
现有技术中,有使用高效液相色谱-三重四级杆/复合线性离子阱质谱,并以甲基睾酮为内标的检测方法快速、同时测定大鼠不同脑区中神经甾体的含量(刘佳,张朦,万有等.高效液相色谱-三重四级杆/复合线性离子阱质谱测定鼠脑组织中神经甾体类化合物[J].分析化学,2015,43(08):1118-1124.);
但发现,根据前述文献的方法试验时发现AP(别孕烯醇酮)的标准溶液不成线性,响应极低;且PREG(孕烯醇酮)和PROG(孕酮)基质效应明显;同时第一次回收率试验结果一般。因此需要考虑改进测定过程中的部分步骤以提高其回收率。
发明内容
本发明针对上述现有技术存在的问题,目的是提供一种高效液相色谱串联质谱法测定神经甾体类激素的方法。
为实现本发明的目的,通过以下技术方案予以实现:
一种高效液相色谱串联质谱法测定神经甾体类激素的方法,包括以下步骤:
(1)标准溶液的配制:称取神经甾体标准品配制成标准溶液;
(2)样品前处理:取待测样品,称重后加入乙腈和MT,涡旋并离心,取上清液;将上清液加入至预先用乙腈清洗过的EMR固相萃取小柱中过滤,得净化后的样品溶液;
(3)高效液相色谱-串联质谱分析:利用高效液相色谱-串联质谱仪分别对标准溶液和样品溶液进行检测分析。
优选地,步骤(3)中,高效液相色谱条件为:
XDB-C18色谱柱;流动相A为乙酸铵溶液,流动相B为甲醇;采用梯度洗脱模式:
0-2.5min时,流动相A:流动相B=40%:60%
2.5-5.5min时,流动相A:流动相B=40%:60%-60%:95%
5.5-7.0min时,流动相A:流动相B=5%:95%
7.0-7.1min时,流动相A:流动相B=5%:40%-95%:60%
7.1-10.0min时,流动相A:流动相B=40%:60%
流速0.5mL/min,进样体积10μL,柱温45℃。
优选地,步骤(3)中,质谱条件为:
离子源:电喷雾离子源;
扫描方式:正电子;
扫描模式:多反应离子监测;
离子化电压:5500V;
气帘气:241.33kpa;
喷雾气:413.7kPa;
辅助加热气:413.7kPa;
离子源温度:100℃。
进一步优选地,所述步骤(2)中,MT的浓度为1μg/mL。
进一步优选地,所述步骤(2)中,离心的条件为15000r/min转速离心4分钟。
进一步优选地,所述步骤(1)中,神经甾体标准品包括脱氧皮质酮、去氢表雄酮、甲基睾酮、孕烯醇酮、孕酮和别孕烯醇酮中的至少一种
本发明通过乙腈沉淀蛋白提取后过EMR小柱的方法去除蛋白等干扰成分,解决AP的标准溶液不成线性,响应极低的问题;同时使得测定的各神经甾体具有较好的回收率。
附图说明
图1-图6分别为实施例一中DOC、DHEA、MT、PREG、PROG、AP的标准曲线;
图7-图12分别为实施例二DOC、DHEA、MT、PREG、PROG、AP的色谱图。
图13为实施例六的AP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。其中,附图仅用于示例性说明,表示的仅是示意图,而非实物图,不能理解为对本专利的限制。
仪器和试剂:
分析纯氨水、色谱级乙酸铵、质谱级甲醇、质谱级乙腈、牛血清白蛋白、氯化钠、分析纯盐酸、标准品别孕烯醇酮、标准品别孕烯醇酮、标准品别孕酮、标准品别脱氧皮质酮、标准品别四氢脱氧皮质酮、标准品别甲基睾酮、标准品别去氢表雄酮;
MCX固相萃取小柱、EMR固相萃取小柱、HLB固相萃取小柱、一次性注射器、一次性滤膜、Thermo Accucore C18 2.6μm 2.1*100mm色谱柱
其余并未列举的仪器、试剂或样品与高效液相色谱-三重四级杆/复合线性离子阱质谱测定鼠脑组织中神经甾体类化合物中(刘佳,张朦,万有等.高效液相色谱-三重四级杆/复合线性离子阱质谱测定鼠脑组织中神经甾体类化合物[J].分析化学,2015,43(08):1118-1124.)所列举的相同或更高精度的相同/替代品。
实施例一样品前处理和标准曲线的绘制
将各标准品用甲醇配制成质量浓度为1mg/mL的标准储备液,并根据需要用甲醇-水(1:1,V/V)稀释成适当质量浓度的混合标准溶液,-20℃保存。
配制5%BSA-0.7%NaCl溶液,加入不同浓度的AP,PREG,DOC,THDOC,PROG,DHEA标准品,浓度范围为0.1~100ng/mL(AP为10~200ng/mL),再加入相同浓度(0.3pg/mL)的内标物MT,振荡混匀后进行液液萃取。加入1.2mL乙酸乙酯-正己烷(9:1,V/V),涡旋5min,于12000r/min下离心5min,将上层有机相转入另一洁净离心管中。氮气吹干,100μL甲醇-水(1:1,V/V,0.1%甲酸)复溶。12000r/min离心15min,上清液供HPLC-MS/MS分析。
结果如图1-至图6所示,依次分别为DOC、DHEA、MT、RPEG、RPOG、AP的标准曲线。除AP外,相关系数r=0.9999;检出限均能达到0.1ng/ml,信噪比>3:1;定量限均能达到0.5ng/ml,信噪比>10:1。
实施例二对比试验色谱分析
鼠脑组织:大鼠采用异氟烷麻醉,断头取血,冰浴分离内侧前额叶皮层(mPFC)、海马(HF)和丘脑腹后核(VP),液氮保存,称重(准确称至0.1mg)。每份组织中加入0.49mL超纯水,10μL15pg/mLMT(甲基睾酮),电动匀浆机匀浆,超声。采用液液萃取法和固相萃取法两种方法处理。
固相萃取法:预先以1mL甲醇、1mL水活化平衡StrataC18柱。将组织匀浆液于12000r/min下离心5min,然后将上清液加入StrataC18柱中;以1mL5%甲醇作为淋洗液,淋洗StrataC18柱。抽至近干后,以1mL甲醇洗脱。收集洗脱液,常温下用N2吹干。100μL甲醇-水(1:1,V/V,0.1%甲酸)复溶。12000r/min离心15min,上清液供UFLC-MS/MS分析。
色谱分析:
AgilentXDB-C18色谱柱(50mm×4.6mm,1.8μm,美国安捷伦公司)。流动相:5mmol/L乙酸铵溶液(A)和甲醇(B);采用梯度洗脱模式:0~2.5min,60%B;2.5~5.5min,60%~95%B;5.5~7.0min,95%B;7.0~7.1min,95%~60%B;7.1~10.0min,60%B。流速0.5mL/min。进样体积10μL;样品室温度为室温;柱温45℃;采用内标法进行定量分析。
MRM色谱结果如图7-12中所示,依次分别为DOC、DHEA、MT、RPEG、RPOG、AP的色谱图,其浓度为浓度为10ng/ml。
质谱条件:
电喷雾离子源(ESI),正离子扫描方式,多反应监测扫描(MRM)模式。离子化电压(IS):5500V,气帘气(CUR):241.33kPa,喷雾气(GS1):413.7kPa,辅助加热气(GS2):413.7kPa,碰撞器(CAD):Medium。由于THDOC的氨加合物物受热容易分解,离子源温度(TEM)设为100℃。
实施例三精密度试验
按照实施例二中的实验方法,以标准物质浓度10ng/ml连续进样6次,计算RSD(相对标准偏差),结果如下表1
表1
DOC | DHEA | MT | PREG | PROG | AP | |
RSD% | 1.3 | 1.2 | 0.6 | 0.9 | 1.4 | 2.5 |
实施例四基质效应试验
由于存在基质效应,标准品在甲醇/水溶液中的质谱响应与其在生物基质中的响应不同,所以应将不同浓度的标准品加入到空白基质中,以制作工作曲线。而神经甾体属于内源性物质,无法得到空白基质,于是选用5%BSA-0.7%NaCl作为空白基质。配制浓度为0.1,0.2,0.5,1,2,510ng/mL的6种神经甾体类化合物的溶液。然后按照实施例二的方法,在标准物质浓度10ng/ml的条件下计算其基质效应,结果如表2所示。
表2
DOC | DHEA | MT | PREG | PROG | AP | |
基质效应% | 110.1 | 91.2 | 105.3 | 60.5 | 69.4 | 108.9 |
实施例五回收率试验
按照实施例二中的实验方法,以标准物质浓度10ng/ml连续独立进样3次,计算回收率,结果如下表3。
表3
单位:% | DOC | DHEA | MT | PREG | PROG | AP |
回收率-1 | 120.1 | 105.4 | 110.1 | 56.3 | 69.0 | / |
回收率-2 | 40.5 | 37.9 | 41.0 | 21.9 | 28.5 | 15.4 |
回收率-3 | 52.5 | 44.8 | 56.2 | 33.7 | 27.9 | 14.5 |
上述实施例一至实施例五可知,若按照《高效液相色谱-三重四极杆/复合线性离子阱质谱测定鼠脑组织中神经甾体类化合物》中的方法重复实验,其存在AP的标准溶液不成线性,响应极低,与论文资料不符;同时PREG和PROG基质效应明显。第一次回收率试验结果一般,但与基质效应实验结果相当,说明该方法或本次实验净化不够,影响较大。因此,本发明针对前述方案的缺陷进行了改进。
实施例六
取大鼠采用异氟烷麻醉,断头取血,冰浴后取脑部,称重约为0.4g。加入1.5ml乙腈+0.2mL的1μg/mLMT,涡旋10min,以15000r/min转速离心4分钟,取上清液,定容至2mL,取1mL样品液,到规格为1mL的EMR固相萃取小柱(用前用1mL乙腈先清洗),过滤到样品瓶。
然后再按实施例二中色谱分析中的步骤进行分析,并以标准物质浓度10ng/ml连续独立进样3次,计算回收率,结果如下表4
表4
单位:% | DOC | DHEA | MT | PREG | PROG | AP |
回收率-1 | 88.8 | 90.0 | 89.4 | 93.5 | 91.5 | 88.1 |
回收率-2 | 90.3 | 92.7 | 90.6 | 92.5 | 90.9 | 90.6 |
回收率-3 | 92.6 | 93.4 | 87.6 | 86.5 | 93.4 | 93.2 |
由表4结果可知,通过乙腈沉淀蛋白提取后过EMR固相萃取小柱的方法,相比使用甲醇沉淀蛋白提取后过HLBC18固相萃取小柱的方法;各神经甾体均能达到较好、较稳定的回收率。证明实施例六的方法能有效的除蛋白等其他干扰成分。但此方法在AP附近有干扰峰,需要使用色谱分离,如图13所示,图中框取部分即为干扰峰。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
Claims (6)
1.一种高效液相色谱串联质谱法测定神经甾体类激素的方法,其特征在于,包括以下步骤:
(1)标准溶液的配制:称取神经甾体标准品配制成标准溶液;
(2)样品前处理:取待测样品,称重后加入乙腈和MT,涡旋并离心,取上清液;将上清液加入至预先用乙腈清洗过的EMR固相萃取小柱中过滤,得净化后的样品溶液;
(3)高效液相色谱-串联质谱分析:利用高效液相色谱-串联质谱仪分别对标准溶液和样品溶液进行检测分析。
2.根据权利要求1所述的高效液相色谱串联质谱法测定神经甾体类激素的方法,其特征在于,步骤(3)中,高效液相色谱条件为:
XDB-C18色谱柱;流动相A为乙酸铵溶液,流动相B为甲醇;采用梯度洗脱模式:
0-2.5min时,流动相A:流动相B=40%:60%
2.5-5.5min时,流动相A:流动相B=40%:60%-60%:95%
5.5-7.0min时,流动相A:流动相B=5%:95%
7.0-7.1min时,流动相A:流动相B=5%:40%-95%:60%
7.1-10.0min时,流动相A:流动相B=40%:60%
流速0.5mL/min,进样体积10μL,柱温45℃。
3.根据权利要求1所述的高效液相色谱串联质谱法测定神经甾体类激素的方法,其特征在于,步骤(3)中,质谱条件为:
离子源:电喷雾离子源;
扫描方式:正电子;
扫描模式:多反应离子监测;
离子化电压:5500V;
气帘气:241.33kpa;
喷雾气:413.7kPa;
辅助加热气:413.7kPa;
离子源温度:100℃。
4.根据权利要求1-3任一所述的高效液相色谱串联质谱法测定神经甾体类激素的方法,其特征在于,所述步骤(2)中,MT的浓度为1μg/mL。
5.根据权利要求1-3任一所述的高效液相色谱串联质谱法测定神经甾体类激素的方法,其特征在于,所述步骤(2)中,离心的条件为15000r/min转速离心4分钟。
6.根据权利要求1-3任一所述的高效液相色谱串联质谱法测定神经甾体类激素的方法,其特征在于,所述步骤(1)中,神经甾体标准品包括脱氧皮质酮、去氢表雄酮、甲基睾酮、孕烯醇酮、孕酮和别孕烯醇酮中的至少一种。
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