Urinary metanephrines by liquid chromatography tandem mass spectrometry: Using multiple quantification methods to minimize interferences in a high throughput method
Urinary metanephrines by liquid chromatography tandem mass spectrometry: Using multiple quantification methods to minimize interferences in a high throughput method
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DOI：
10.1016/j.jchromb.2011.10.005
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发表时间：
2011-12-01
影响因子：
3
通讯作者：
Frank, Elizabeth L.
中科院分区：
文献类型：
--
作者：
Clark, Zlatuse D.;Frank, Elizabeth L.
尿中肾上腺素的测定常被用于嗜铬细胞瘤的鉴别诊断和监测。虽然已经开发了许多方法，但干扰是常见的，并且阻碍了大多数可用的测定。本研究描述了一种可靠的高通量LC-MS/MS方法的开发、验证和实施，用于测量尿液中的间甲肾上腺素和去甲间甲肾上腺素。使用96孔格式的混合模式阳离子交换吸附剂上的固相萃取，从进行酸水解的尿液样品中分离出美坦利和去甲美坦利。将提取物直接进样至Restek全氟苯基色谱柱上，并在0.2%甲酸/5%甲醇中进行等度分离，梯度清洗步骤为50%甲醇。使用API 3200三重四极杆质谱仪以正离子模式电喷雾电离完成检测。以多反应监测模式采集数据。监测了间甲萘醌及其氘代内标物的三个跃迁;监测了去甲萘醌及其氘代内标物的两个跃迁。使用两种定量方法来解决代谢物干扰而不降低通量。方法的线性范围为15,000 nmol/L，检出限为2.5 nmol/L，定量限为10 nmol/L。运行内，两种分析物的日间和总不精密度值均等于或低于1.9%、2.5%和2.7%。该方法与我们以前使用的GC-MS方法相关性良好。进样至进样时间为6 min。用于测量尿液标本中的间甲肾上腺素和去甲肾上腺素的经验证的LC-MS/MS方法于2010年8月投入使用，并且表现非常好。(C)2011爱思唯尔有限公司版权所有。
Determination of urinary metanephrines is requested frequently for the differential diagnosis and monitoring of pheochromocytoma. Although numerous methods have been developed, interferences are common and hinder most available assays. This study describes the development, validation and implementation of a reliable high-throughput LC-MS/MS method for the measurement of metanephrine and normetanephrine in urine. Metanephrine and normetanephrine were isolated from urine samples subjected to acid hydrolysis using solid phase extraction on a mixed mode cation exchange sorbent in 96-well format. The extracts were injected directly onto a Restek perfluorophenyl column and separated isocratically in 0.2% formic acid in 5% methanol with a gradient cleanout step to 50% methanol. Detection was accomplished using an API 3200 triple quadrupole mass spectrometer with electrospray ionization in positive mode. Data were acquired in multiple reaction monitoring mode. Three transitions were monitored for metanephrine and its deuterated internal standard; two transitions were monitored for normetanephrine and its deuterated internal standard. Two quantification methods were used to address metanephrine interferences without reducing throughput. The method was linear to 15,000 nmol/L The limits of detection and quantification were 2.5 and 10 nmol/L, respectively. Within run, between-day and total imprecision values were at or below 1.9%, 2.5% and 2.7% for both analytes. The method correlated well with our previously used GC-MS method. Injection-to-injection time was 6 min. The validated LC-MS/MS method for measurement of metanephrine and normetanephrine in urine specimens was placed into service in August 2010 and has performed exceptionally well. (C) 2011 Elsevier B.V. All rights reserved.