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Sensitive Quantitative Analysis Of C-peptide In Human Plasma By 2-dimensional Liquid Chromatography-mass Spectrometry Isotope-dilution Assay.
E. Rogatsky, B. Balent, Gayotri Goswami, V. Tomuta, H. Jayatillake, Greg Cruikshank, Louis Vele, D. Stein
Published 2006 · Chemistry, Medicine
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BACKGROUND Isotope-dilution assays (IDAs) are well established for quantification of metabolites or small drug molecules in biological fluids. Because of their increased specificity, IDAs are an alternative to immunoassays for measuring C-peptide. METHODS We evaluated a 2-dimensional liquid chromatography-mass spectrometry (2D LC/MS) IDA method. Sample preparation was by off-line solid-phase extraction, and C-peptide separation was performed on an Agilent 1100 2D LC system with a purification method based on high-pressure switching between 2 high-resolution reversed-phase columns. Because of the low fragmentation efficiency of C-peptide, multiple-reaction monitoring analysis was omitted and selective-ion monitoring mode was chosen for quantification. Native and isotope-labeled ([M+18] and [M+30]) C-peptides were monitored in the +3 state at m/z 1007.7, 1013.7, and 1017.7. RESULTS The assay was linear (r(2) = 0.9995), with a detection limit of 300 amole (1 pg) on column. Inter- and intraday CVs for C-peptide were < or =2%. Comparison with an established polyclonal-based RIA showed high correlation (r = 0.964). Plasma concentrations of total C-peptide measured by RIA were consistently higher than by IDA LC/MS, consistent with the higher specificity of IDAs compared with immunoassays. CONCLUSIONS The 2D LC/MS IDA approach eliminates matrix effects, enhancing assay performance and reliability, and has a detection limit 100-fold lower than any previously reported LC/MS method. Isotope-labeled C-peptide(s) can be clearly differentiated from endogenous C-peptide by the difference in m/z ratio, so that both peptides can be quantified simultaneously. The method is highly precise, robust, and applicable to pharmacokinetic detection of plasma peptides.
This paper references
Quantitative determination of the polypeptide motilin in rat plasma by externally calibrated liquid chromatography/electrospray ionization mass spectrometry.
D. C. Delinsky (2004)
Effects of dopexamine in comparison with fenoterol on carbohydrate, fat and protein metabolism in healthy volunteers
W. Geisser (2003)
Insulin Biosynthesis: Evidence for a Precursor
D. Steiner (1967)
Quantitative study of insulin secretion and clearance in normal and obese subjects.
K. Polonsky (1988)
Assays for Insulin, Proinsulin(S) and C-Peptide
P. Clark (1999)
Development of an Isotope Dilution Assay for Precise Determination of Insulin, C-peptide, and Proinsulin Levels in Non-diabetic and Type II Diabetic Individuals with Comparison to Immunoassay*
A. D. Kippen (1997)
Quantitation of the large polypeptide glucagon by protein precipitation and LC/MS.
D. C. Delinsky (2004)
Recommendations by the Commission on Physico-Chemical Symbols and Terminology under the International Union of Pure and Applied Chemistry (IUPAC), 1955.
J. A. Christiansen (1957)
Radioactive and Stable Isotope Tracers in Biomedicine
R. Bramlet (1993)
WHO international reference reagents for human proinsulin and human insulin C-peptide.
A. Bristow (1988)
A NOVEL SECRETORY PRODUCT OF THE RAT PANCREATIC BETA CELL PRODUCED BY TRUNCATION OF PROINSULIN CONNECTING PEPTIDE IN SECRETORY GRANULES*
C. Bruce Verchere (1996)
Quantitative analysis of urinary C-peptide by liquid chromatography-tandem mass spectrometry with a stable isotopically labelled internal standard.
C. Fierens (2000)
Direct sensitive quantitative lC/MS analysis of C-peptide from human urine by two dimensional reverse phase/reverse phase high-performance liquid chromatography.
E. Rogatsky (2006)
SLIDE HOLDERS FOR OVERCOMING A DEFECT IN ATTACHABLE MECHANICAL STAGES.
H. W. Graybill (1923)
Linco Research, Inc
Linco Research (2005)
Anhydrous Protein Ions
C. S. Hoaglund-Hyzer (1999)
A Stable Isotope Dilution Assay for the In Vivo Determination of Insulin Levels in Humans by Mass Spectrometry
R. Stöcklin (1997)
C. B. Verchere (1996)
Matrix effect in the quantitative analysis of urinary C-peptide by liquid chromatography/mass spectrometry.
C. Fierens (2000)
Quantitative determination of chloramphenicol in milk powders by isotope dilution liquid chromatography coupled to tandem mass spectrometry.
P. Guy (2004)
Receptor binding and biological potency of several split forms (conversion intermediates) of human proinsulin. Studies in cultured IM-9 lymphocytes and in vivo and in vitro in rats.
D. Peavy (1985)
Application of a C-peptide electrospray ionization-isotope dilution-liquid chromatography-tandem mass spectrometry measurement procedure for the evaluation of five C-peptide immunoassays for urine.
C. Fierens (2003)
Compendium of Chemical Terminology
A. Wilkinson (1997)
A mass spectrometric method for quantitation of intact insulin in blood samples.
S. M. Darby (2001)
Accurate Assessment of β-Cell Function: The Hyperbolic Correction
R. Bergman (2002)
Proinsulin C-peptide and its C-terminal pentapeptide: degradation in human serum and Schiff base formation with subsequent CO2 incorporation
E. Melles (2003)
Insulin sensitivity, insulin secretion, and abdominal fat: the Insulin Resistance Atherosclerosis Study (IRAS) Family Study.
L. Wagenknecht (2003)
Hepatic removal of insulin in normal man: dose response to endogenous insulin secretion.
R. Eaton (1983)
C-Peptide as a Measure of the Secretion and Hepatic Extraction of Insulin: Pitfalls and Limitations
K. Polonsky (1984)
Quantitation of Human Pancreatic Beta-cell Function by Immunoassay of C-Peptide in Urine
D. Horwitz (1977)
This paper is referenced by
Human C-peptide Quantitation by LC-MS Isotope-Dilution Assay in Serum or Urine Samples
Alexander V Stoyanov (2013)
Implementing a Reference Measurement System for C-Peptide: Successes and Lessons Learned.
R. Little (2017)
Implementing a Reference Measurement System for C-Peptide: An Addendum.
R. Little (2017)
C‐peptide microheterogeneity in type 2 diabetes populations
Paul E. Oran (2010)
Quantitative Peptidomics Using Reductive Methylation of Amines.
Sayani Dasgupta (2018)
Two-step ion-exchange chromatographic purification combined with reversed-phase chromatography to isolate C-peptide for mass spectrometric analysis.
Kuanysh Z. Kabytaev (2016)
Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry.
I. van den Broek (2008)
Comparison between a linear ion trap and a triple quadruple MS in the sensitive detection of large peptides at femtomole amounts on column.
Bilgin Vatansever (2010)
Standardization of insulin and C-peptide – A status report
K. V. Uytfanghe (2010)
Multidimensional LC-MS/MS enables simultaneous quantification of intact human insulin and five recombinant analogs in human plasma.
E. Chambers (2014)
Liquid chromatography coupled to tandem mass spectrometry for the quantitative bioanalysis of bioactive and potential biomarker peptides
I. Broek (2010)
Comprehensive Proteome Analysis of Human Smooth Muscle Cells by 2D-HPLC-ESI-MS/MS
Yongqian Zhang (2011)
Liquid chromatography-tandem mass spectrometry approach for quantification of mucins from sputum using 13C,15N-labeled peptides as internal standards.
Claes Lindberg (2013)
Advances in the quantitation of therapeutic insulin analogues by LC-MS/MS.
M. Blackburn (2013)
Quantification of Intact and Truncated Stromal Cell-Derived Factor-1α in Circulation by Immunoaffinity Enrichment and Tandem Mass Spectrometry
W. Wang (2014)
Targeted Quantification of Peptide Biomarkers: A Case Study of Amyloid Peptides
L. Dillen (2017)
Biochemical view on "Homocysteine and metabolic syndrome: From clustering to additional utility in prediction of coronary heart disease".
E. Sertoğlu (2015)
A simple purification protocol for the detection of peptide hormones in the hemolymph of individual insects by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Sandy Fastner (2007)
Standardization of C-peptide measurements.
R. Little (2008)
Highly sensitive and quantitative analysis of polyeptides using a new gradient system based on an abrupt change in adsorption of polypeptide to the reversed-phase column packing.
R. Goda (2007)
Quantification of serum C-peptide by isotope-dilution liquid chromatography-tandem mass spectrometry: enhanced detection using chemical modification and immunoaffinity purification.
T. Kinumi (2014)
IEF‐based multidimensional applications in proteomics: Toward higher resolution
Alexander V Stoyanov (2012)
Proteomic applications of protein quantification by isotope-dilution mass spectrometry
V. Mayya (2006)
Development of a pretreatment method for amyloid beta-protein analysis based on the effect of acetic acid on the dissolution of plasma polypeptides.
R. Goda (2008)
Simultaneous Monitoring of Multiple Transitions in Mass Spectrometric Analysis Improves Limit of Detection for Low Abundance Substances inComplex Biological Samples
Dmitriy Shin (2013)
A step toward simplicity for a complex analyte.
C. Bystrom (2011)
An immunoaffinity liquid chromatography-tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum.
M. F. Ocaña (2010)
International comparison of C-peptide measurements.
H. Wiedmeyer (2007)
Application of Fused-Core Particle Column in Two Dimensional Reversed Phase - Reversed Phase LC/MS Analysis of Biological Samples. Impact of Extra-Column Volume
E. Rogatsky (2012)
Targeted Proteomics Studies: Design, Development and Translation of Mass Spectrometric Immunoassays for Diabetes and Kidney Disease
Paul E. Oran (2011)
Factors determining insulin requirements in women with type 1 diabetes mellitus during pregnancy: a review
N. Achong (2014)
A sensitive electrochemiluminescent biosensor based on AuNP-functionalized ITO for a label-free immunoassay of C-peptide.
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