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This Article Full Text Full Text (PDF) 063081.Supplemental Data All Versions of this Article: clinchem.2005.063081v1 52/5/872    most recent Submit an electronic Letter to the Editor about this paper Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Rogatsky, E. Articles by Stein, D. T. Search for Related Content PubMed PubMed Citation Articles by Rogatsky, E. Articles by Stein, D. T. Related Collections Endocrinology and Metabolism

Sensitive Quantitative Analysis of C-Peptide in Human Plasma by 2-Dimensional Liquid Chromatography–Mass Spectrometry Isotope-Dilution Assay

¹ Department of Medicine and ² General Clinical Research Center at Albert Einstein College of Medicine of Yeshiva University, Bronx, NY.

^aAddress correspondence to this author at: Department of Medicine, Division of Endocrinology and Metabolism, Albert Einstein College of Medicine, 1300 Morris Park Ave., Room G47, Bronx, NY 10461. Fax 718-430-2446; e-mail dstein{at}aecom.yu.edu.

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 (r² = 0.9995), with a detection limit of 300 amole (1 pg) on column. Inter- and intraday CVs for C-peptide were 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.

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				R. R. Little, C. L. Rohlfing, A. L. Tennill, R. W. Madsen, K. S. Polonsky, G. L. Myers, C. J. Greenbaum, J. P. Palmer, E. Rogatsky, and D. T. Stein Standardization of C-Peptide Measurements Clin. Chem., June 1, 2008; 54(6): 1023 - 1026. [Abstract] [Full Text] [PDF]

				H.-M. Wiedmeyer, K. S. Polonsky, G. L. Myers, R. R. Little, C. J. Greenbaum, D. E. Goldstein, and J. P. Palmer International Comparison of C-Peptide Measurements Clin. Chem., April 1, 2007; 53(4): 784 - 787. [Abstract] [Full Text] [PDF]