Département de Chimie, Bureau International des Poids et Mesures (BIPM), Pavillon de Breteuil, 92312, Sèvres Cedex, France.
Division of Chemical Metrology & Analytical Science, National Institute of Metrology, Beijing, 100029, People's Republic of China.
Anal Bioanal Chem. 2018 Aug;410(20):5059-5070. doi: 10.1007/s00216-018-1155-y. Epub 2018 Jun 4.
Peptides are an increasingly important group of biomarkers and pharmaceuticals. The accurate purity characterization of peptide calibrators is critical for the development of reference measurement systems for laboratory medicine and quality control of pharmaceuticals. The peptides used for these purposes are increasingly produced through peptide synthesis. Various approaches (for example mass balance, amino acid analysis, qNMR, and nitrogen determination) can be applied to accurately value assign the purity of peptide calibrators. However, all purity assessment approaches require a correction for structurally related peptide impurities in order to avoid biases. Liquid chromatography coupled to high resolution mass spectrometry (LC-hrMS) has become the key technique for the identification and accurate quantification of structurally related peptide impurities in intact peptide calibrator materials. In this study, LC-hrMS-based methods were developed and validated in-house for the identification and quantification of structurally related peptide impurities in a synthetic human C-peptide (hCP) material, which served as a study material for an international comparison looking at the competencies of laboratories to perform peptide purity mass fraction assignments. More than 65 impurities were identified, confirmed, and accurately quantified by using LC-hrMS. The total mass fraction of all structurally related peptide impurities in the hCP study material was estimated to be 83.3 mg/g with an associated expanded uncertainty of 3.0 mg/g (k = 2). The calibration hierarchy concept used for the quantification of individual impurities is described in detail. Graphical abstract ᅟ.
肽是一类日益重要的生物标志物和药物。准确鉴定肽标准物质的纯度对于建立医学实验室参考测量系统和药物质量控制至关重要。这些目的所使用的肽越来越多地通过肽合成来生产。各种方法（例如质量平衡、氨基酸分析、qNMR 和氮测定）可用于准确赋值肽标准物质的纯度。然而，所有纯度评估方法都需要对结构相关肽杂质进行校正，以避免偏差。液相色谱-高分辨质谱联用（LC-hrMS）已成为鉴定和准确量化完整肽标准物质中结构相关肽杂质的关键技术。在这项研究中，开发并在内部验证了基于 LC-hrMS 的方法，用于鉴定和量化合成人 C 肽（hCP）材料中的结构相关肽杂质。该材料被用作国际比较研究的研究材料，旨在考察实验室进行肽纯度质量分数赋值的能力。使用 LC-hrMS 鉴定、确认并准确量化了超过 65 种杂质。hCP 研究材料中所有结构相关肽杂质的总质量分数估计为 83.3mg/g，相对扩展不确定度为 3.0mg/g（k=2）。详细描述了用于量化个别杂质的校准层次概念。
