Background: Urinary 5-hydroxyindoleacetic acid (5-HIAA) is a first line investigation for gastrointestinal neuroendocrine tumours (NETs) that secrete serotonin. It also has clinical utility for monitoring disease progression and therapeutic response.

Aim: To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for urinary 5-HIAA that incorporates a supported liquid extraction (SLE) and 13C labelled internal standard.

Methods: Samples were diluted in ammonium acetate containing a 13C labelled internal standard (5-Hydroxyindole-3a,4,5,6,7,7a-13C6-3-acetic acid). SLE was performed followed by chromatographic separation using the 2.1 x 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-HIAA and 198.2 > 152.4 m/z for 13C-5-HIAA.

Results: A well-defined 5-HIAA peak was observed at 0.8 min with a run time of 2.4 mins. 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 (EQA) 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 hrs post-extraction with minimal carryover.

Conclusion: 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 EQA material and is in routine service for clinical samples.

背景：尿液 5-羟基吲哚乙酸 (5-HIAA) 是分泌血清素的胃肠道神经内分泌肿瘤 (NET) 的一线研究。它还具有监测疾病进展和治疗反应的临床效用。

目的：开发和验证一种用于尿液 5-HIAA 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 (SLE) 和 13C 标记的内标。

方法：样品在含有 13C 标记内标（5-Hydroxyindole-3a,4,5,6,7,7a-13C6-3-乙酸）的乙酸铵中稀释。使用 2.1 x 30 mm CORTECS® UPLC® T3 色谱柱进行 SLE，然后进行色谱分离。质谱检测 (Waters Xevo TQ-XS) 在电喷雾正离子模式下使用离子对 192.3> 146.4 m/z（定量器）和 192.3>118.4 m/z（定性器）进行 5-HIAA 和 198.2 > 152.4 m/z 13C-5-HIAA。

结果：在 0.8 分钟时观察到明确定义的 5-HIAA 峰，运行时间为 2.4 分钟。该测定与 382 µmol/L 呈线性 (r2 >0.99)，定量下限为 5.3 µmol/L (CV <15%)。对 29 个外部质量保证 (EQA) 样本的分析表明，我们的方法与 UKNEQAS 方法的平均值（4.7% 正偏差）之间的一致性很好。测定内和测定间精密度在可接受的范围内，并且该测定在提取后 96 小时内保持稳定，残留最少。

结论：我们开发了一种稳健的半自动萃取 LC-MS/MS 方法，与现有的劳动密集型 HPLC 方法相比，它提供了改进的运行时间和性能。该方法快速、精确，与 UKNEQAS EQA 材料具有良好的一致性，并已用于临床样品的常规服务。