Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the “sticky” character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5–40 μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials.

中文翻译：

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改进的LC-MS / MS方法定量临床样品中的hepcidin-25

基于质谱的方法通过从其他天然存在的N截短的亚型（hepcidin-20，-22，-24）中选出具有生物活性的25个残基肽，在主要铁代谢调节剂hepcidin的定量中起着至关重要的作用。似乎在铁稳态中没有活性。然而，由于肽的“粘性”特征和缺乏合适的标准品，在铁调素的MS分析中出现了一些困难。在此，我们建议在测试几种类型的小瓶用于制备储备溶液和血清样品以进行同位素稀释液相色谱-串联质谱（ID-）的测试后，使用氨基和氟硅烷化的自动进样器小瓶来减少铁调素与实验室玻璃器皿表面的相互作用。 LC-MS / MS）。此外，我们研究了两种样品制备策略和两种色谱分离条件，目的是开发一种LC-MS / MS方法对血清样品中hepcidin-25进行灵敏可靠的定量。将基于常规酸性流动相的色谱分离与一种新方法进行了比较，该新方法涉及用含0.1％氨的高pH值的溶剂分离hepcidin-25。两种方法均使用相同的hepcidin-25校正剂在两种LC-MS / MS方法的实验室内比较中应用于临床样品，结果具有良好的相关性。最后，我们推荐使用基于LC-MS / MS的定量方法，其动态范围为0.5–40μg/ L，用于评估人血清中的hepcidin-25，该方法使用基于TFA的流动相和硅烷化玻璃瓶。