Background Most first-tier newborn screening (NBS) biomarkers are evaluated by a 2-min flow injection analysis coupled to tandem mass spectrometry (FIA-MS/MS) assay. The absence of separation prior to MS/MS analysis can lead to false positives and inconclusive results due to interferences by nominal isobars and isomers. Therefore, many presumptive positive specimens require confirmation by a higher specificity second-tier assay employing separations, which require additional time and resources prior to patient follow-up. Methods A 3.2-mm punch was taken from dried blood spot (DBS) specimens and extracted using a solution containing isotopically labeled internal standards for quantification. Analyses were carried out in positive mode using a commercially available microfluidic capillary electrophoresis (CE) system coupled to a high-resolution mass spectrometer (HRMS). Results The CE-HRMS platform quantified 35 first- and second-tier biomarkers from a single injection in <2-min acquisition time, thus, successfully multiplexing first- and second-tier NBS for over 20 disorders in a single DBS punch. The CE-HRMS platform resolved problematic isobars and isomers that affect first-tier FIA-MS/MS assay specificity, while achieving similar quantitative results and assay linearity. Conclusions Our CE-HRMS assay is capable of multiplexing first- and second-tier NBS biomarkers into a single assay with an acquisition time of <2 min. Such an assay would reduce the volume of false positives and inconclusive specimens flagged for second-tier screening.

中文翻译：

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使用基于高通量芯片的毛细管电泳与高分辨率质谱联用在一次检测中结合第一层和第二层新生儿筛查

背景 大多数第一层新生儿筛查 (NBS) 生物标志物通过 2 分钟流动注射分析与串联质谱 (FIA-MS/MS) 测定进行评估。由于标称等压线和异构体的干扰，在 MS/MS 分析之前没有分离会导致假阳性和不确定的结果。因此，许多推定阳性标本需要通过采用分离的更高特异性第二层测定来确认，这需要在患者随访之前额外的时间和资源。方法 从干血斑 (DBS) 样本中取出一个 3.2 毫米的穿孔器，并使用含有同位素标记的内标的溶液进行定量提取。使用与高分辨率质谱仪 (HRMS) 耦合的市售微流体毛细管电泳 (CE) 系统以正模式进行分析。结果 CE-HRMS 平台在不到 2 分钟的采集时间内从一次注射中量化了 35 个一​​级和二级生物标志物，因此，在一次 DBS 冲床中成功地将一级和二级 NBS 多路复用用于 20 多种疾病。CE-HRMS 平台解决了影响一级 FIA-MS/MS 测定特异性的有问题的等压线和异构体，同时实现了相似的定量结果和测定线性。结论 我们的 CE-HRMS 测定能够将一级和二级 NBS 生物标志物多路复用到单一测定中，采集时间小于 2 分钟。