CN112763602A - 一种血清中甾体激素的快速检测方法 - Google Patents
一种血清中甾体激素的快速检测方法 Download PDFInfo
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Abstract
本发明涉及激素检测技术领域,公开了一种血清中甾体激素的快速检测方法,包括激素校准品制备、样本制备、超高效液相色谱分离、串联四级杆质谱检测、单点定量分析等步骤:该检测方法创新性的使用同位素标记的[13C,2H3]‑甲氧胺(盐酸盐)衍生激素校准品,未经同位素标记的甲氧胺衍生血清样本,二者衍生产物按一定体积比混合,经超高效液相色谱分离,三重四极杆串联质谱检测,单点法定量,快速准确的检测血清样本中13种甾体激素。
Description
技术领域
本发明涉及激素检测技术领域,具体涉及一种血清中甾体激素的快速检测方法。
背景技术
甾体激素是人体内最重要的内源性代谢物种类之一,在维持第二性征、调控基因转录、调节内分泌与免疫功能等方面发挥着至关重要的作用。对整体的甾体激素代谢网络的定量测定对于研究疾病的发生发展机制和个体对疾病的应答具有重要的作用。
目前,内源性甾体激素的检测方法主要有免疫分析、气质联用分析(GC-MS)以及液质联用分析(LC-MS)技术。免疫分析方法能够对单个激素进行高灵敏检测,但是由于基质干扰严重并且抗体的批次差异较大导致其特异性、重复性和准确度均较差。GC-MS可对多个甾体激素同时进行检测,且大大提高了检测特异性,但是GC-MS的检测灵敏度有限且样品预处理复杂制约了GC-MS在常规临床实验室中的广泛应用。LC-MS方法能够同时对多个甾体激素进行高灵敏度特异检测,是一种对整体甾体激素代谢网络进行高通量分析的理想技术。
不经过衍化采用LC-MS方法是目前较为常用的方法,具有前处理较为简单,方法稳定可靠的有点,但是由于不同的甾体激素在离子化过程中的差异较大,许多甾体激素在电喷雾离子源中的离子化效率很低,造成其检测灵敏度很差。化学衍生化技术是一种分析化学常用的技术,用于提高化合物的紫外、荧光和质谱响应。在LC-MS分析中,衍生化试剂通过与目标化合物的某一化学基团反应,提高目标化合物在液相色谱柱上的保留能力和质谱分析的离子化效率,提高检测灵敏度的同时,有效避免了在多种离子化模式和离子化极性的切换。
针对已有衍生化方法的文献报道进行检索,检索中国发明专利一种基于化学衍生的尿液中甾体激素的检测方法(授权公告号为CN103698414B),其主要针对尿液中甾体激素的检测,未见报道血液中甾体激素的检测。
发明内容
针对现有技术存在的不足,本发明的目的在于提供一种血清中甾体激素的快速检测方法,本检测方法创新性的使用同位素标记的[13C,2H3]-甲氧胺(盐酸盐)衍生激素校准品,未经同位素标记的甲氧胺衍生血清样本,二者衍生产物按一定体积比混合,经超高效液相色谱分离,三重四极杆串联质谱检测,单点法定量,快速准确的检测血清样本中13种甾体激素。
为了实现上述目的,本发明提供如下技术方案:
一种血清中甾体激素的快速检测方法,该检测方法包括以下步骤:激素校准品制备,样本制备,超高效液相色谱分离,串联四级杆质谱检测血清中多种激素物质(包括睾酮、孕酮、17-α-羟孕酮、皮质醇、11-脱氧皮质醇、皮质酮、雄烯二酮、脱氧皮质酮、二氢睾酮、脱氢表雄酮、孕烯醇酮、雌酮、脱氢表雄酮硫酸酯),单点定量分析;
同时,本申请涉及的13种甾体激素与医院常规性早熟6项检查项目具有很强的相关性,都是针对血清样本的检测;相比于尿液,血清中甾体激素的改变能够被更早检测到。
激素校准品制备、样本制备:
1)离心管中依次加入沉淀剂、待测血清或激素混合标准溶液,涡旋振荡;
2)离心管中加入萃取剂萃取,涡旋,离心,收集上清液;
3)将步骤2)中上清液冻干,冻干后进行衍生,激素混合标准溶液衍生时,采用同位素标记的衍生试剂,待测血清衍生时,采用未用同位素标记的衍生试剂;衍生结束后再次冻干;
4)冻干结束后,加入复溶液,盖紧离心管盖子,涡旋,离心,取上清液,得到血清样本的衍生产物或校准品衍生产物;
超高效液相色谱分离与串联四级杆质谱检测:
5)将上述血清样本的衍生产物、校准品衍生产物混合,涡旋混匀后,在色谱柱上梯度洗脱分离,进入质谱系统检测;
单点定量分析:
6)单点定量分析,在前期验证线性相关性良好的基础上,采用校准曲线的一个浓度点,利用浓度与峰面积成正比的关系,计算出血清中激素的含量。
作为优选,步骤1)中沉淀剂为质量体积浓度10%硫酸锌水溶液,沉淀剂与待测血清或激素混合标准溶液的体积比为1:7-10;
步骤1)中涡旋振荡时长为2min;
步骤2)中萃取液为乙酸乙酯,萃取液体积是待萃取混合液体积的2-3倍。
作为优选,步骤2)中离心条件为4℃条件下以13000rpm/s速度离心,且涡旋5min,离心5min。
作为优选,同位素标记的衍生试剂为[13C,2H3]-甲氧胺(盐酸盐)吡啶溶液,未用同位素标记的衍生试剂为甲氧胺(盐酸盐)吡啶溶液;质量浓度均为20mg/mL。
作为优选,步骤3)中衍生方法为向萃取冻干后的离心管内加入浓度为衍生试剂,涡旋30s,室温超声15min,37℃下衍生90min。
作为优选,步骤4)中复溶液为体积浓度30%甲醇/水,涡旋30s后,离心5min。
作为优选,步骤5)中血清样本的衍生产物、校准品衍生产物混合按照体积比为1:1的比例混合。
作为优选,步骤5)中色谱柱为Agilent Proshell 120EC-C18 3.0×50mm 2.7μm,梯度洗脱中流动相A为0.1%甲酸/水溶液,流动相B为0.1%甲酸/乙腈溶液,
梯度洗脱条件为流动相B的体积比为:0~1.5min为30%~45%,1.5~2.5min为45%~55%,2.5~3.0min为55%~100%,3.0~4.0min为100%~100%,4.0~4.1min为100%~30%,4.1~4.5min为30%~30%;
流速为0.4ml/mim,柱温为50℃,进样量为7.5μL。
作为优选,甾体激素包括但不限于睾酮、孕酮、17-α-羟孕酮、11-脱氧皮质醇、皮质酮、雄烯二酮、脱氢表雄酮、孕烯醇酮、脱氧皮质酮、二氢睾酮、雌酮、皮质醇、脱氢表雄酮硫酸酯。
与现有技术相比,本发明的有益效果是:
本发明方法采用同位素标记的衍生试剂衍生校准品,不再需要使用每个检测物质的同位素内标进行定性定量,同位素内标价格昂贵,极大的降低了检测成本;甾体激素经衍生后检测,有效的提高了检测灵敏度;在前处理过程中不需要加入同位素内标,同时可用单点进行定量,不需要校准曲线进行定量,节省了校准曲线配制、前处理及检测时间,有效提高样本分析通量,进一步降低检测成本。
附图说明
图1为睾酮的甲氧胺衍生产物及[13C,2H3]-甲氧胺衍生产物的LC-MS图谱。
图2为孕酮的甲氧胺衍生产物及[13C,2H3]-甲氧胺衍生产物的LC-MS图谱。
图3为17-α-羟孕酮的甲氧胺衍生产物及[13C,2H3]-甲氧胺衍生产物的LC-MS图谱。
图4为皮质醇的甲氧胺衍生产物及[13C,2H3]-甲氧胺衍生产物的LC-MS图谱。
图5为11-脱氧皮质醇的甲氧胺衍生产物及[13C,2H3]-甲氧胺衍生产物的LC-MS图谱。
图6为皮质酮的甲氧胺衍生产物及[13C,2H3]-甲氧胺衍生产物的LC-MS图谱。
图7为脱氧皮质酮的甲氧胺衍生产物及[13C,2H3]-甲氧胺衍生产物的LC-MS图谱。
图8为二氢睾酮的甲氧胺衍生产物及[13C,2H3]-甲氧胺衍生产物的LC-MS图谱。
图9为脱氢表雄酮的甲氧胺衍生产物及[13C,2H3]-甲氧胺衍生产物的LC-MS图谱。
图10为孕烯醇酮的甲氧胺衍生产物及[13C,2H3]-甲氧胺衍生产物的LC-MS图谱。
图11为雌酮的甲氧胺衍生产物及[13C,2H3]-甲氧胺衍生产物的LC-MS图谱。
图12为脱氢表雄酮硫酸酯的甲氧胺衍生产物及[13C,2H3]-甲氧胺衍生产物的LC-MS图谱。
图13为雄烯二酮的甲氧胺衍生产物及[13C,2H3]-甲氧胺衍生产物的LC-MS图谱。
具体实施方式
一种检测血清中13种甾体激素的快速质谱检测方法,包括以下步骤:
1)配置激素混合标准溶液,13种激素为睾酮、孕酮、17-α-羟孕酮、皮质醇、11-脱氧皮质醇、皮质酮、雄烯二酮、脱氧皮质酮、二氢睾酮、脱氢表雄酮、孕烯醇酮、雌酮、脱氢表雄酮硫酸酯,其浓度见表1所示:
表1
2)激素校准品制备:移取20μL10%硫酸锌水溶液于1.5mL离心管内,移取150μL 13种激素混合校准溶液加入该离心管内,涡旋振荡2min,再移取600μL乙酸乙酯加入该离心管内,涡旋振荡5min,离心5min,移取500μL上清液至干净的1.5mL离心管,于真空冷冻干燥机内冻干;
冻干结束后,于离心管内加入100μL 20mg/mL的[13C,2H3]-甲氧胺盐酸盐吡啶溶液,涡旋30s,室温超声15min,37℃条件下衍生90min,衍生结束后于真空冷冻干燥机内冻干;
冻干结束后,加入80μL 30%甲醇/水复溶,涡旋30s,离心5min,取上清液,得到校准品衍生产物;
3)样品制备:移取20μL10%硫酸锌水溶液于1.5mL离心管内,移取150μL血清样本加入该离心管内,涡旋振荡2min,再移取600μL乙酸乙酯加入该离心管内,涡旋振荡5min,离心5min,移取500μL上清液至干净的1.5mL离心管,于真空冷冻干燥机内冻干;
冻干结束后,于离心管内加入100μL 20mg/mL的甲氧胺盐酸盐吡啶溶液,涡旋30s,室温超声15min,37℃条件下衍生90min,衍生结束后于真空冷冻干燥机内冻干;
冻干结束后,加入80μL 30%甲醇/水复溶,涡旋30s,离心5min,取上清液,得到血清样本衍生产物;
4)超高效液相色谱分离:将步骤2)得到的校准品衍生产物和步骤3)得到的血清样本衍生产物按体积比1:1混合,涡旋混匀,应用超高效液相色谱系统,在反相核壳填料色谱柱上,流动相梯度洗脱分离,
具体色谱条件为:色谱柱为Agilent Proshell 120EC-C18 3.0×50mm 2.7μm,流动相A为0.1%甲酸/水溶液,流动相B为0.1%甲酸/乙腈溶液,梯度洗脱条件见表2,流速为0.4ml/mim,柱温为50℃,进样量为7.5μL;
表2
时间(min) | 流动相A(%) | 流动相B(%) |
0 | 70 | 30 |
1.5 | 55 | 45 |
2.5 | 45 | 55 |
3.0 | 0 | 100 |
4.0 | 0 | 100 |
4.1 | 70 | 30 |
5.5 | 70 | 30 |
13种激素的[13C,2H3]-甲氧胺衍生产物和13种激素的甲氧胺衍生产物在色谱柱上分离,同一种激素的[13C,2H3]-甲氧胺衍生产物和甲氧胺衍生产物保留时间一致,后续根据质谱信息进行定性定量,13种激素衍生产物保留时间见表3。
表3
序号 | 激素 | 保留时间(min) |
1 | 睾酮 | 2.65 |
2 | 孕酮 | 3.46 |
3 | 17-α-羟孕酮 | 2.58 |
4 | 皮质醇 | 1.44 |
5 | 11-脱氧皮质醇 | 2.20 |
6 | 皮质酮 | 1.71 |
7 | 雄烯二酮 | 2.52 |
8 | 脱氧皮质酮 | 2.43 |
9 | 二氢睾酮 | 2.84 |
10 | 脱氢表雄酮 | 2.45 |
11 | 孕烯醇酮 | 3.51 |
12 | 雌酮 | 2.42 |
13 | 脱氢表雄酮硫酸酯 | 1.48 |
5)串联四级杆质谱检测:色谱柱流出的[13C,2H3]-甲氧胺衍生产物和13种激素的甲氧胺衍生产物进入质谱系统检测,使用正离子模式,特定的离子源参数和MRM参数,检测26种激素的衍生产物;质谱检测条件为ESI源,正离子模式,离子源参数见表4,MRM参数见表5。
表4
表5
6)单点定量分析:在前期验证线性相关性良好的基础上,采用校准曲线的一个浓度点(具体浓度见表1),利用浓度与峰面积成正比的关系,计算出血清中激素的含量。
将该方法应用于试剂血清样本检测,选取20例实际血清样本,具体检测结果见表6。
表6(1)
表6(2)
注:上述数据为5次检测的平均值。
由上可知:采用本检测方法能够快速准确的检测血清样本中睾酮、孕酮、17-α-羟孕酮、皮质醇、11-脱氧皮质醇、皮质酮、雄烯二酮、脱氧皮质酮、二氢睾酮、脱氢表雄酮、孕烯醇酮、雌酮及脱氢表雄酮硫酸酯13种甾体激素,检测灵敏度较高;在前处理过程中不需要加入同位素内标,同时可用单点进行定量,不需要校准曲线进行定量,节省了校准曲线配制、前处理及检测时间,有效提高样本分析通量,进一步降低检测成本。
Claims (10)
1.一种血清中甾体激素的快速检测方法,其特征在于,包括以下步骤:
1)离心管中依次加入沉淀剂、待测血清或激素混合标准溶液,涡旋振荡;
2)离心管中加入萃取剂萃取,涡旋,离心,收集上清液;
3)将步骤2)中上清液冻干,冻干后进行衍生,激素混合标准溶液衍生时,采用同位素标记的衍生试剂,待测血清衍生时,采用未用同位素标记的衍生试剂;衍生结束后再次冻干;
4)冻干结束后,加入复溶液,盖紧离心管盖子,涡旋,离心,取上清液,得到血清样本的衍生产物或校准品衍生产物;
5)将上述血清样本的衍生产物、校准品衍生产物混合,涡旋混匀后在色谱柱上梯度洗脱分离,进入质谱系统检测;
6)单点定量分析,计算出血清中激素的含量。
2.根据权利要求1所述的一种血清中甾体激素的快速检测方法,其特征在于,步骤1)中沉淀剂为质量体积浓度10%硫酸锌水溶液,沉淀剂与待测血清或激素混合标准溶液的体积比为1:7-10;
步骤2)中萃取液为乙酸乙酯,萃取液体积是待萃取混合液体积的2-3倍。
3.根据权利要求2所述的一种血清中甾体激素的快速检测方法,其特征在于,步骤2)中离心条件为4℃条件下以13000rpm/s速度离心。
4.根据权利要求1所述的一种血清中甾体激素的快速检测方法,其特征在于,同位素标记的衍生试剂为[13C,2H3]-甲氧胺(盐酸盐)吡啶溶液,未用同位素标记的衍生试剂为甲氧胺(盐酸盐)吡啶溶液;质量浓度均为20mg/mL。
5.根据权利要求4所述的一种血清中甾体激素的快速检测方法,其特征在于,步骤3)中衍生方法为向萃取冻干后的离心管内加入浓度为衍生试剂,涡旋,室温超声,37℃下衍生90min。
6.根据权利要求1所述的一种血清中甾体激素的快速检测方法,其特征在于,步骤4)中复溶液为体积浓度30%甲醇/水。
7.根据权利要求1所述的一种血清中甾体激素的快速检测方法,其特征在于,步骤5)中血清样本的衍生产物、校准品衍生产物混合按照体积比为1:1的比例混合。
8.根据权利要求7所述的一种血清中甾体激素的快速检测方法,其特征在于,步骤5)中色谱柱为Agilent Proshell 120EC-C18 3.0×50mm 2.7μm,
梯度洗脱中流动相A为0.1%甲酸/水溶液,流动相B为0.1%甲酸/乙腈溶液。
9.根据权利要求8所述的一种血清中甾体激素的快速检测方法,其特征在于,梯度洗脱条件为流动相B的体积比为:0~1.5min为30%~45%,1.5~2.5min为45%~55%,2.5~3.0min为55%~100%,3.0~4.0min为100%~100%,4.0~4.1min为100%~30%,4.1~4.5min为30%~30%;
流速为0.4ml/mim,柱温为50℃,进样量为7.5μL。
10.根据权利要求1所述的一种血清中甾体激素的快速检测方法,其特征在于,甾体激素包括但不限于睾酮、孕酮、17-α-羟孕酮、11-脱氧皮质醇、皮质酮、雄烯二酮、脱氢表雄酮、孕烯醇酮、脱氧皮质酮、二氢睾酮、雌酮、皮质醇、脱氢表雄酮硫酸酯。
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