尿5-羟基吲哚乙酸(5-HIAA)是分泌5-羟色胺的胃肠道神经内分泌肿瘤的一线研究。它还具有用于监测疾病进展和治疗反应的临床效用。
开发和验证尿中5-羟基吲哚乙酸的液相色谱-串联质谱(LC-MS/MS)方法,该方法结合了支持的液体萃取和13C标记的内标。
样品在含有13C标记的内标(5-羟基吲哚-3a,4,5,6,7,7a-13C6-3-乙酸)。进行支持的液体提取,然后使用2.1×30mmCORTECS®UPLC®T3柱进行色谱分离。质谱检测(WatersXevoTQ-XS)以电喷雾正模式进行,使用5-羟基吲哚乙酸的跃迁192.3>146.4m/z(定量)和192.3>118.4m/z(限定符)和13C-5-HIAA的跃迁198.2>152.4m/z。
在0.8分钟观察到明确定义的5-羟基吲哚乙酸峰,运行时间为2.4分钟。该测定线性(r2>0.99)至382μmol/L,定量下限为5.3µmol/L(CV<15%)。对29个外部质量保证样品的分析显示,我们的方法与UKNEQAS方法平均值(正偏差4.7%)之间具有良好的一致性。测定内和测定间的精确度在可接受的范围内,并且该测定在提取后96小时内稳定,残留最少。
我们开发了一种具有半自动提取功能的强大的LC-MS/MS方法,与现有的方法相比,该方法提供了改进的运行时间和性能,劳动密集型,HPLC方法。方法很快,精确,与UKNEQAS外部质量保证材料表现出良好的一致性,并且正在为临床样品提供常规服务。
Urinary 5-hydroxyindoleacetic acid (5-HIAA) is a first-line investigation for gastrointestinal neuroendocrine tumours that secrete serotonin. It also has clinical utility for monitoring disease progression and therapeutic response.
To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for urinary 5-hydroxyindoleacetic acid that incorporates a supported liquid extraction and 13C-labelled internal standard.
Samples were diluted in ammonium acetate containing a 13C-labelled internal standard (5-hydroxyindole-3a,4,5,6,7,7a-13C6-3-acetic acid). Supported liquid extraction was performed followed by chromatographic separation using the 2.1 × 30 mm CORTECS® UPLC® T3 column. Mass spectrometry detection (Waters Xevo TQ-XS) was performed in electrospray positive mode using the transitions 192.3 > 146.4 m/z (quantifier) and 192.3 > 118.4 m/z (qualifier) for 5-hydroxyindoleacetic acid and 198.2 > 152.4 m/z for 13C-5-HIAA.
A well-defined 5-hydroxyindoleacetic acid peak was observed at 0.8 min with a run time of 2.4 min. The assay was linear (r2 > 0.99) to 382 µmol/L, with a lower limit of quantification of 5.3 µmol/L (CV <15%). Analysis of 29 external quality assurance samples showed good agreement between our method and the UKNEQAS method mean (4.7% positive bias). The intra- and inter-assay precision was within acceptable limits, and the assay was stable up to 96 h postextraction with minimal carryover.
We have developed a robust LC-MS/MS method with semi-automated extraction that offers an improved run time and performance over the existing, labour-intensive, HPLC method. The method was quick, precise, showed good agreement with UKNEQAS external quality assurance material and is in routine service for clinical samples.