Standardization of C-Peptide Measurements Randie R. Little, 1* Curt L. Rohlfing, 1 Alethea L. Tennill, 1 Richard W. Madsen, 2 Kenneth S. Polonsky, 3 Gary L. Myers, 4 Carla J. Greenbaum, 5 Jerry P. Palmer, 6 Eduard Rogatsky, 7 and Daniel T. Stein 7 BACKGROUND: C-peptide is a marker of insulin secre- tion in diabetic patients. We assessed within- and be- tween-laboratory imprecision of C-peptide assays and determined whether serum calibrators with values as- signed by mass spectrometry could be used to harmo- nize C-peptide results. METHODS: We sent 40 different serum samples to 15 laboratories, which used 9 different routine C-peptide assay methods. We also sent matched plasma samples to another laboratory for C-peptide analysis with a ref- erence mass spectrometry method. Each laboratory analyzed 8 of these samples in duplicate on each of 4 days to evaluate within- and between-day impreci- sion. The same 8 samples were also used to normalize the results for the remaining samples to the mass spec- trometry reference method. RESULTS: Within- and between-run CVs ranged from 2% to 10% and from 2% to 18%, respectively. Normalizing the results with serum samples signifi- cantly improved the comparability among laboratories and methods. After normalization, the differences among laboratories in mean response were no longer statis- tically significant (P = 0.24), with least-squares means of 0.93–1.02. CONCLUSIONS: C-peptide results generated by different methods and laboratories do not always agree, espe- cially at higher C-peptide concentrations. Within- laboratory imprecision also varied, with some methods giving much more consistent results than others. These data show that calibrating C-peptide measurement to a reference method can increase comparability between laboratories. © 2008 American Association for Clinical Chemistry The C-peptide concentration provides an accurate assessment of residual -cell function in humans and has become an important marker of insulin se- cretion in patients with diabetes (1, 2 ). C-peptide assessment is also useful in the diagnosis of insuli- noma/endogenous hyperinsulinemia (3). The Dia- betes Control and Complications Trial has shown that higher C-peptide concentrations are associated with improved glycosylated hemoglobin (HbA1c) concentrations, less hypoglycemia, and less retinop- athy and nephropathy (4). A stable C-peptide con- centration is therefore being used as a measurable endpoint in immunomodulatory trials for type 1 diabetes (5, 6 ). C-peptide may also play a role in preventing and reversing some complications of type 1 diabetes (7–9 ). Our previous study showed that the among- laboratory imprecision in C-peptide measurements is considerable and that although calibration with pure C-peptide standards (WHO IRR 84/510) did not suc- cessfully improve comparability, patient samples could successfully be used to calibrate assays and re- duce imprecision (10 ). Two isotope-dilution liquid chromatography–mass spectrometry (LC-MS) 8 meth- ods for measuring C-peptide have been described. One method uses 2-dimensional LC (11 ), and the other uses tandem MS (12 ) to improve the detection limit and specificity of the analysis. In addition, a small method-comparison study showed that normalization to an LC-MS reference method could improve the comparability of results (12 ). We describe the use of the 2-dimensional LC-MS isotope-dilution method to assign reference values to plasma samples, which were then transferred to corresponding serum-sample cali- brators in 9 different clinical laboratory methods in 15 laboratories. 1 Department of Pathology & Anatomical Sciences, University of Missouri School of Medicine, Columbia, MO; 2 Department of Statistics, University of Missouri School of Medicine, Columbia, MO; 3 Department of Medicine, Washington University School of Medicine, St. Louis, MO; 4 Centers for Disease Control and Prevention, Division of Environmental Health Laboratory Sciences, Centers for Environmental Health, Chamblee, GA; 5 Benaroya Research Institute, Seattle, WA; 6 University of Washington and VA Medical Center, Seattle, WA; 7 Depart- ment of Medicine and GCRC Analytical Core Laboratory, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY. * Address correspondence to this author at: Diabetes Diagnostic Laboratory, M767, Department of Pathology & Anatomical Sciences, University of Missouri School of Medicine, 1 Hospital Dr., Columbia, MO 65212. Fax 573-884-8823; e-mail LittleR@health.missouri.edu. Received November 28, 2007; accepted March 5, 2008. Previously published online at DOI: 10.1373/clinchem.2007.101287 8 Nonstandard abbreviation: LC-MS, liquid chromatography–mass spectrometry. Clinical Chemistry 54:6 1023–1026 (2008) Endocrinology and Metabolism 1023