Biomaterial Database

Isotope-dilution liquid chromatography-tandem mass spectrometry for sensitive quantification of human insulin in serum using derivatization-technique. 2017

Yohei Sakaguchi, and Tomoya Kinumi, and Akiko Takatsu

Bio-medical Standards Group, Research Institute for Material and Chemical Measurement, National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba C-3, Ibaraki 305-8563, Japan. Electronic address: yohei-sakaguchi@aist.go.jp.

An isotope-dilution mass spectrometry (IDMS) method for measuring insulin levels in human serum was developed using C-terminal-derivatization method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The carboxyl groups of Glu-C-cleavage products were derivatized with 1-(2-pyrimidinyl)piperazine to increase MS/MS sensitivity and IDMS quantification, resulting in increases in LC-MS/MS peak areas of derivatized Glu-C-cleavage products of human insulin by ∼23-(A5-17 peptide) to 49-fold(B14-21 peptide), respectively, as compared with results observed in the absence of derivatization. Separation was achieved on a C18 column by gradient elution at 0.3 mL/min, with a mobile phase composed of 0.1% formic acid in acetonitrile and water. Validation studies of target peptides (B1-13 peptide and B14-21 peptide) revealed a linear response in the range of 0.05 ng/mL to 10 ng/mL (regression coefficient, r2 = 0.9987 and 0.9988, respectively), a relative standard deviation within and between days of <8.6%, and spike and recovery test results indicating mean recoveries ranging from 100.2% to 106.6%. Comparison with an established commercial immunoassay showed high correlation (r2 = 0.9943 and 0.9944, B1-13 peptide and B14-21 peptide, respectively) at serum concentrations of between 0.20 ng/mL and 1.51 ng/mL. These findings suggested that this IDMS-based approach was able to quantify human serum insulin with high sensitivity and precision in the reference interval and indicated a potential for determining serum-insulin reference-measurement procedures to allow traceable measurement.

UI	MeSH Term	Description	Entries
D007553	Isotope Labeling	Techniques for labeling a substance with a stable or radioactive isotope. It is not used for articles involving labeled substances unless the methods of labeling are substantively discussed. Tracers that may be labeled include chemical substances, cells, or microorganisms.	Isotope Labeling, Stable,Isotope-Coded Affinity Tagging,Isotopically-Coded Affinity Tagging,Affinity Tagging, Isotope-Coded,Affinity Tagging, Isotopically-Coded,Isotope Coded Affinity Tagging,Labeling, Isotope,Labeling, Stable Isotope,Stable Isotope Labeling,Tagging, Isotope-Coded Affinity,Tagging, Isotopically-Coded Affinity
D010455	Peptides	Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are considered to be larger versions of peptides that can form into complex structures such as ENZYMES and RECEPTORS.	Peptide,Polypeptide,Polypeptides
D002851	Chromatography, High Pressure Liquid	Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed.	Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D003903	Deuterium	The stable isotope of hydrogen. It has one neutron and one proton in the nucleus.	Deuterons,Hydrogen-2,Hydrogen 2
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D012697	Serine Endopeptidases	Any member of the group of ENDOPEPTIDASES containing at the active site a serine residue involved in catalysis.	Serine Endopeptidase,Endopeptidase, Serine,Endopeptidases, Serine
D053719	Tandem Mass Spectrometry	A mass spectrometry technique using two (MS/MS) or more mass analyzers. With two in tandem, the precursor ions are mass-selected by a first mass analyzer, and focused into a collision region where they are then fragmented into product ions which are then characterized by a second mass analyzer. A variety of techniques are used to separate the compounds, ionize them, and introduce them to the first mass analyzer. For example, for in GC-MS/MS, GAS CHROMATOGRAPHY-MASS SPECTROMETRY is involved in separating relatively small compounds by GAS CHROMATOGRAPHY prior to injecting them into an ionization chamber for the mass selection.	Mass Spectrometry-Mass Spectrometry,Mass Spectrometry Mass Spectrometry,Mass Spectrometry, Tandem
D061385	Insulins	Peptide hormones that cause an increase in the absorption of GLUCOSE by cells within organs such as LIVER; MUSCLE and ADIPOSE TISSUE. During normal metabolism insulins are produced by the PANCREATIC BETA CELLS in response to increased GLUCOSE. Natural and chemically-modified forms of insulin are also used in the treatment of GLUCOSE METABOLISM DISORDERS such as DIABETES MELLITUS.