Stoppacher N, Josephs R D, Daireaux A, Choteau T, Westwood S, Wielgosz R I
Bureau International des Poids et Mesures (BIPM), Département de Chimie, Pavillon de Breteuil, 92312, Sèvres Cedex, France.
Rapid Commun Mass Spectrom. 2015 Sep 30;29(18):1651-60. doi: 10.1002/rcm.7261.
The prohormone angiotensin I (ANG I) [amino acid sequence: DRVYIHPFHL] and other structurally related peptide hormones play an essential role in the regulation of the water and electrolyte balance in the human body as well as blood pressure. ANG I is a biomarker for hypertension and diabetes. Therefore, well-characterized pure reference materials and comparable and SI-traceable analytical characterization methods are required to establish reference measurement systems (RMS) for laboratory medicine.
Two analytical characterization methods based on liquid chromatography/mass spectrometry (LC/MS) systems with electrospray ionization have been developed and validated in-house. Both high-resolution MS (hrMS) and hybrid-tandem MS/MS were used for the identification and quantification of the major structurally related peptide impurities of ANG I. The impurities were quantified by use of external calibrations with original impurity standards. Mass fraction impurity values and corresponding expanded measurement uncertainties were calculated.
Five structurally related degradation products were detected as major impurities in a 'pure' ANG I material. The peptides ANG (2-10) [RVYIHPFHL], ANG II [DRVYIHPF] and three ANG I isomers [DRVYLHPFHL, DRVYIHPFHI and DRVYLHPFHI] were identified and corresponding mass fraction values calculated that range from 0.66 to 4.86 mg/g.
The mass fraction values for the major related peptide impurities in the ANG I material obtained with both LC/hrMS and LC/MS/MS systems are in excellent agreement. This study emphasizes the importance of mass spectrometric techniques for application to mass balance approaches for mass fraction value and uncertainty assignment of impurities in 'pure' substance reference materials for peptides.
激素原血管紧张素I（ANG I）[氨基酸序列：DRVYIHPFHL]及其他结构相关的肽类激素在人体水和电解质平衡以及血压调节中起着至关重要的作用。ANG I是高血压和糖尿病的生物标志物。因此，需要特性明确的纯参考物质以及可比较且可溯源至国际单位制（SI）的分析表征方法，以建立检验医学的参考测量系统（RMS）。
已开发并在内部验证了两种基于配备电喷雾电离的液相色谱/质谱（LC/MS）系统的分析表征方法。高分辨率质谱（hrMS）和混合串联质谱/质谱（MS/MS）均用于鉴定和定量ANG I主要的结构相关肽杂质。通过使用原始杂质标准品进行外标法对杂质进行定量。计算质量分数杂质值和相应的扩展测量不确定度。
在一种“纯”ANG I物质中检测到五种结构相关的降解产物作为主要杂质。鉴定出肽ANG（2 - 10）[RVYIHPFHL]、血管紧张素II [DRVYIHPF]以及三种ANG I异构体[DRVYLHPFHL、DRVYIHPFHI和DRVYLHPFHI]，并计算出相应的质量分数值，范围为0.66至4.86 mg/g。
使用LC/hrMS和LC/MS/MS系统获得的ANG I物质中主要相关肽杂质的质量分数值高度一致。本研究强调了质谱技术在应用于肽类“纯”物质参考材料中杂质质量分数值和不确定度赋值的质量平衡方法中的重要性。
