CN117554536B - 一种环境水样中15种甲状腺激素的同时分析方法 - Google Patents
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Abstract
本发明公开了一种环境水样中15种甲状腺激素的同时分析方法,本发明首次提出利用强疏水性硅胶基质C18吸附剂增强反相保留甲状腺激素碘取代苯环疏水结构,保证不同碘取代程度和脱酸脱氨分析物同时充分保留,从而提高固相萃取富集浓缩回收率,结合超高效液相色谱‑质谱联用技术(UPLC‑MS/MS)色谱有效分离和质谱正/负离子电离模式同时使用,实现环境水样中15种不同酸/碱性、碘取代个数和空间结构甲状腺激素的高灵敏同时检测。本发明通过对方法学验证,线性、准确度、精密度均满足要求,可用于环境水样中多种不同结构痕量甲状腺激素的同时分析。
Description
技术领域
本发明属于检测技术领域,是一种基于强疏水吸附剂增强碘取代苯环结构反相保留从而实现环境水样中15种不同结构甲状腺激素的同时分析方法。
背景技术
甲状腺激素(THs)是一种由甲状腺分泌的激素,几乎可以作用于人体全部细胞,在营养代谢、体格生长、大脑发育、完善神经及心血管功能等方面起着至关重要的作用。这类物质经人类和动物天然排泄以及人工合成药物的使用进入环境,可能对生态健康产生重要危害,特别是对鱼类和两栖类的早期发育和变态过程。研究发现四碘甲状腺原氨酸(T4)在几十个ng/L水平就能对变态前蝌蚪甲状腺激素相关基因转录产生影响、影响变态,甚至导致死亡;水产养殖中经常报道的仔鱼骨骼发育异常可能跟暴露3,3′,5-三碘甲状腺原氨酸(T3)和T4相关。因此,亟需建立分析方法调查和评估该类新污染物的污染水平及环境风险。
T3和T4是临床上检测的常规指标,血清中浓度水平一般在几十个μg/L,前处理主要是去除血清中蛋白的干扰;而环境水样中THs往往在很低的ng/L、甚至pg/L水平,且干扰检测的环境基质成分极其复杂,比如广泛存在的溶解有机质除了包含多肽结构特征的蛋白,还含有与THs可能发现相互作用的多酚、羟基、醛酮等多种结构特征的干扰成分。以往适合几百微升血清样品的前处理方法很难应用于几百毫升、基质更复杂环境水样的浓缩与净化。
另外,T3和T4 可能发生脱碘、脱羧、脱氨和醚键断裂反应,生成仍然具有生物活性但是酸/碱性、亲/疏水性、空间结构不同的代谢转化产物。通过对现有环境水样THs分析方法的文献报道进行检索,并没有一种可以同时检测环境水样中超过5种(T4,T3, rT3,T2和rT2)、特别是包括脱羧/脱氨产物的方法。具有广泛保留性的Oasis HLB吸附剂能够一定程度保留环境水样中二到四碘取代THs。由于THs结构中碘原子的空间位阻作用,HLB骨架上二乙烯基苯疏水结构对THs吸附能力较弱,THs羧基与N-乙烯吡咯烷酮亲水结构氢键作用可能是主要保留机理,因此,THs脱酸产物难以与其他THs同时从环境水样中富集分析。
发明内容
为了解决上述问题,本发明首次提出利用强疏水性硅胶基质C18 吸附剂增强反相保留THs碘取代苯环疏水结构,保证不同碘取代程度和脱酸脱氨分析物同时充分保留,从而提高固相萃取富集浓缩回收率,并结合超高效液相色谱-质谱联用技术(UPLC-MS/MS)色谱有效分离和质谱正/负离子电离模式同时使用,实现环境水样中15种THs的同时分析方法,为全面评估THs环境污染及其引起的生态健康风险提供支撑。
本发明涉及的15种THs名称为:T4、T3、rT3、T2、rT2、MIT、DIT、T0、T1、rT1、T1AM、T2AM、TA4、TA3、TA2。
为了达到上述目的,本发明采用如下技术方案:
一种环境水样中15种甲状腺激素的同时分析方法,包括如下步骤:
S1:建立环境水样中THs的固相萃取方法,选取合适的固相萃取柱,优化洗脱溶剂,提高回收率并降低干扰;
S2:建立与优化15种THs的质谱参数,选取合适的质谱条件;
S3:选择色谱柱,优化流动相组成和比例,提高15种THs的分离效果和检测灵敏度。
优选的,作为一个较佳的实施方式,所述S1中固相萃取方法的优化:根据15种目标THs的结构特征及可能的保留机理,选取回收率最好的固相萃取柱及洗脱溶剂。所述固相萃取柱种类包括亲水亲脂平衡聚合物吸附剂Oasis HLB(500 mg, 6cc, Waters),极性中等硅胶基质吸附剂Sep-Pak NH2(500 mg, 6cc, Waters)和强疏水性硅胶基质吸附剂SEP-PakC18固相萃取柱(500 mg, 6cc, Waters);所述洗脱溶剂选取甲醇和0.1%氨水甲醇溶液。
优选的,作为一个较佳的实施方式,所述S2中质谱参数的选择与优化:根据15种目标THs的结构及离子化效果确定正/负离子采集模式和特征离子对;优化锥孔电压、碰撞能量,提高特征离子对质谱响应强度和稳定性。
优选的,作为一个较佳的实施方式,所述S3色谱柱选择HPH-C18色谱柱(100 mm×2.1 mm, 2.7µm, Agilent);所述流动相水相选取纯水和0.1%乙酸水溶液,流速在仪器和色谱柱适配的范围内选择0.2、0.3和0.4 mL/min,色谱柱的柱温依据THs性质选择45 ℃以下。
优选的,作为一个较佳的实施方式,本发明首先确定了能够同时富集萃取环境水样中15种目标THs的固相萃取柱及洗脱溶剂,进一步建立与优化每一种THs的质谱检测特征离子和条件,结合色谱分离条件优化,实现了15种不同结构THs的同时分析。
与现有技术相比,本发明的有益效果为:
(1)本发明首次提出基于强疏水吸附剂增强碘取代苯环结构反相保留,保证不同碘取代程度和脱酸脱氨产物同时充分保留,从而提高固相萃取回收率。
(2)本发明采用UPLC-MS/MS检测技术,基于目标THs的基线色谱分离和正/负离子模式分段扫描,实现了15种不同酸/碱性、碘取代个数和空间结构THs的高灵敏同时检测。
(3)本发明通过对方法学验证,线性、准确度、精密度均满足要求,可用于环境水样中多种不同结构痕量THs的同时分析。
附图说明
图1为15种甲状腺激素的结构图;
图2为UPLC-MS/MS同时检测15种不同结构THs的色谱图。
具体实施方式
以下将以检测环境水样中甲状腺激素浓度为例对本发明的技术方案进行更为详细的说明,但这些实施例不对本发明构成任何限制。
一、实施例1:地表水中甲状腺激素浓度的检测
1、仪器与试剂:
超高效液相色谱串联质谱联用仪(Waters公司),包括ACQUITY超高效液相系统,TQ-S四级杆质谱;氮吹仪;涡旋振荡器(Vortex-Genie 2),固相萃取装置(SUPELCOVISIPREP 24TMDL)。
甲醇(LC/MS级)、乙酸(LC/MS级)、氨水(LC/MS级)、Milli-Q水。
甲状腺激素标准品及同位素内标:包括表1中的T4、T3、rT3、rT2、T2、T1、rT1、T0、MIT、DIT、13C6-T4、T2AM、T1AM购买于Toronto Research Chemicals (Downsview, ON,Canada), T4标准品的纯度大于99.5%,T3、13C6-T4和T1AM标准品的纯度大于98%;rT3标准品的纯度大于96%,rT2、T2、T1和T2AM标准品的纯度大于97%;rT1、T0、MIT和DIT标准品的纯度大于95%。13C6-T3、13C6-T2、13C6-MIT标准品购买于Cambridge Isotope Laboratories(Andover,MA, USA)纯度均大于95%、97%、98%。TA4、TA3、TA2标准品购买于广州佳途科技股份有限公司,TA4纯度大于 94 %;TA3纯度大于97%;TA2纯度大于99%。
表1 15种甲状腺激素的质谱参数和检出限(LOQ)
;
2、样品前处理:
取500 mL环境水样,经玻璃纤维滤膜过滤后,利用三种固相萃取柱对添加相同浓度标准溶液的样品进行富集浓(n=3),在各自绝对回收率最高的条件下进行对比(表2),最终选择强疏水性硅胶基质C18吸附剂,洗脱溶剂为甲醇;固相萃取整个过程,首先用6 mL二氯甲烷、6 mL甲醇及12 mL超纯水活化,过滤后的水样中添加5 ng同位素内标,将水样以5-10 mL/min的流速通过,用10 mL超纯水淋洗并吹干。最后用6 mL甲醇洗脱、氮气吹干,用甲醇复溶至200 µL,进入仪器进行分析。
表2 三种不同保留机理固相萃取柱萃取15种目标甲状腺激素的绝对回收率对比
;
3、超高效液相色谱串联质谱(UPLC-MS/MS)检测:
(1)质谱分析条件:采用电喷雾离子源(ESI),TA4、TA3和TA2为负离子检测,其他THs为正离子检测。质谱检测采用多反应监测模式(MRM),毛细管电压3 kV;脱溶剂气温度600 °C;离子源温度150 °C;脱溶剂气体流量900 L/Hr;碰撞气流量0.15 mL/min。TA4、TA3和TA2在ESI-MS/MS在负离模式下运行,选取的平均参数为:毛细管电压2.5 kV;脱溶剂气温度200 °C;离子源温度150 °C;脱溶剂气体流量800 L/Hr;碰撞气流量0.15 mL/min。主要的质谱参数见表1。
(2)UPLC液相条件:色谱柱选择HPH-C18色谱柱,有机相选择甲醇,水相对比纯水和0.1%乙酸水溶液条件下15种THs的色谱分离效果及质谱响应灵敏度,确定选择0.1%乙酸水溶液。最终流动相采用B(甲醇)和A(0.1%乙酸水溶液),梯度条件为:0 min,90%A;0-3 min,90%A;3-6 min,90%-50%A;6-10 min,50-10%A;10-10.5 min,10%-0%A;10.5-12 min,0%A;12-12.1 min,0%-90%A;12.1-16 min,90%A;经过对比不同流速下的柱压、峰宽、物质洗脱情况,最终流速为0.3 mL/min;柱温为35°C,进样量为2 μL。
4、方法的考核参数及结果:
本发明采用高效液相色谱质谱联用的方法,通过保留时间和两对离子对对每一种类固醇激素进行确认,再根据标准品峰面积进行定量分析,并用对应的同位素内标校正目标物质在样品前处理过程和仪器分析过程中的损失,并且弥补进样过程中针与针之间的差异。具体参数如下:
(1)特异性分析:针对三个方面进行特异性评估。首先,针对标准溶液的色谱图进行分离度评估,以确认物质(尤其是同分异构体)间是否能有效分离。其次,利用空白水样(超纯水)模拟完整的样品处理过程,包括使用相同的仪器、试剂、药品和玻璃器皿。最后,向空白水样(超纯水)中加入已知浓度的目标分析物,与样品一同分析,检查是否存在干扰物。通过液相色谱条件的优化,本方法显示出良好的分离效果,特别是对于难以分离的同分异构体。在实验中,空白样品与标准溶液相比未出现干扰信号。
(2)标准曲线与最低定量限的测定:在空白基质中添加1 ng/L,5 ng/L,10 ng/L,50 ng/L,100ng/L的标准溶液,进样量为2 μL进行UPLC-MS/MS分析,并对各物质峰面积与内标峰面积的比值(Y)和其浓度(X)进行线性回归分析,获得标准曲线。结果表明,物质的浓度与所测得相对内标的峰面积呈现良好的线性关系,相关系数(γ)基本大于0.99。依据信噪比(S/N)计算得各甲状腺激素的最低定量限(LOQ)为0.3-26 ng/L。
(3)回收率和重复性的测定:将一定10 ng/L浓度的标准品加入空白基质中,按照样品前处理及仪器分析方法进行检测,并计算该方法的回收率,以每个浓度重复测定6次的结果计算方法重现性,结果表明,甲状腺激素回收率为96-112%,相对标准偏差为(RSD)3.3-11%。
综上所述,仅为本发明具体实施方式,但本发明的保护范围并不限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围内,因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (2)
1.一种环境水样中15种甲状腺激素的同时分析方法,其特征在于,包括如下步骤:
S1 样品前处理
取环境水样,利用强疏水性硅胶基质吸附剂SEP-Pak C18固相萃取柱,500 mg, 6cc,Waters,富集环境样品,首先用6 mL二氯甲烷、6 mL甲醇及12 mL超纯水活化,过滤后的水样中添加5 ng同位素内标,将水样以5-10 mL/min的流速通过,用10 mL超纯水淋洗并吹干;最后用6 mL甲醇洗脱、氮气吹干,用甲醇复溶至200 µL,进入仪器进行分析;
S2超高效液相色谱串联质谱检测
质谱分析条件:采用电喷雾离子源ESI,四碘甲状腺乙酸、三碘甲状腺乙酸和二碘甲状腺乙酸为负离子检测,其他甲状腺激素为正离子检测;质谱检测采用多反应监测模式MRM,毛细管电压3 kV;脱溶剂气温度600 °C;离子源温度150 °C;脱溶剂气体流量900 L/Hr;碰撞气流量0.15 mL/min;四碘甲状腺乙酸、三碘甲状腺乙酸和二碘甲状腺乙酸在ESI-MS/MS在负离子模式下运行,选取的平均参数为:毛细管电压2.5 kV;脱溶剂气温度200 °C;离子源温度150 °C;脱溶剂气体流量800 L/Hr;碰撞气流量0.15 mL/min;
UPLC液相条件:色谱柱选择HPH-C18色谱柱,有机相选择甲醇,水相对比纯水和0.1%乙酸水溶液条件下15种甲状腺激素的色谱分离效果及质谱响应灵敏度,确定选择0.1%乙酸水溶液;最终流动相采用B:甲醇,A:0.1%乙酸水溶液,梯度条件为:0 min,90%A;0-3 min,90%A;3-6 min,90%-50%A;6-10 min,50-10%A;10-10.5 min,10%-0%A;10.5-12 min,0%A;12-12.1 min,0%-90%A;12.1-16 min,90%A;经过对比不同流速下的柱压、峰宽、物质洗脱情况,最终流速为0.3 mL/min;柱温为35°C,进样量为2 μL;
所述15种甲状腺激素为:四碘甲状腺原氨酸、3,3′,5-三碘甲状腺原氨酸、3,3′,5′-三碘甲状腺氨酸、3,3′-二碘甲状腺氨酸、3,5-二碘-L-甲状腺氨酸、一碘酪氨酸、二碘酪氨酸、甲状腺氨酸、3-碘-L-甲状腺氨酸、3′-碘-L-甲状腺氨酸、3,5-二碘甲状腺乙胺、3-碘甲状腺乙胺、四碘甲状腺乙酸、三碘甲状腺乙酸、二碘甲状腺乙酸。
2. 根据权利要求1所述的环境水样中15种甲状腺激素的同时分析方法,其特征在于,所述流动相水相选取纯水和0.1%乙酸水溶液,流速在仪器和色谱柱适配的范围内选择0.2、0.3和0.4 mL/min,色谱柱的柱温依据甲状腺激素性质选择45 ℃以下。
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