title: Development of SI-traceable C-peptide certified reference material NMIJ CRM 6901-a using isotope-dilution mass spectrometry-based amino acid analyses.
abstract: A certified reference material (CRM) is a higher-order calibration material used to enable a traceable analysis. This paper describes the development of a C-peptide CRM (NMIJ CRM 6901-a) by the National Metrology Institute of Japan using two independent methods for amino acid analysis based on isotope-dilution mass spectrometry. C-peptide is a 31-mer peptide that is utilized for the evaluation of β-cell function in the pancreas in clinical testing. This CRM is a lyophilized synthetic peptide having the human C-peptide sequence, and contains deamidated and pyroglutamylated forms of C-peptide. By adding water (1.00 ± 0.01) g into the vial containing the CRM, the C-peptide solution in 10 mM phosphate buffer saline (pH 6.6) is reconstituted. We assigned two certified values that represent the concentrations of total C-peptide (mixture of C-peptide, deamidated C-peptide, and pyroglutamylated C-peptide) and C-peptide. The certified concentration of total C-peptide was determined by two amino acid analyses using pre-column derivatization liquid chromatography-mass spectrometry and hydrophilic chromatography-mass spectrometry following acid hydrolysis. The certified concentration of C-peptide was determined by multiplying the concentration of total C-peptide by the ratio of the relative area of C-peptide to that of the total C-peptide measured by liquid chromatography. The certified value of C-peptide (80.7 ± 5.0) mg/L represents the concentration of the specific entity of C-peptide; on the other hand, the certified value of total C-peptide, (81.7 ± 5.1) mg/L can be used for analyses that does not differentiate deamidated and pyroglutamylated C-peptide from C-peptide itself, such as amino acid analyses and immunochemical assays.
authors.affiliation: Bio-Medical Standard Section, National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan. t.kinumi@aist.go.jp
authors.forename: Tomoya
authors.initials: T
authors.lastname: Kinumi
authors.forename: Mari
authors.initials: M
authors.lastname: Goto
authors.forename: Sakae
authors.initials: S
authors.lastname: Eyama
authors.forename: Megumi
authors.initials: M
authors.lastname: Kato
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kasama
authors.forename: Akiko
authors.initials: A
authors.lastname: Takatsu
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D002096:C-Peptide / Q000032:analysis*; D002138:Calibration; D006801:Humans; D007201:Indicator Dilution Techniques; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards*; D008969:Molecular Sequence Data; D012015:Reference Standards
chemical_list: D000596:Amino Acids; D002096:C-Peptide