Quantitative Amino Acid Analysis in Insulin and C-Peptide Assays

To the Editor:Standardization of peptide assays is challenging. Most commercial assays are standardized against WHO reference materials. Unfortunately, many of these materials are provided ( a ) with values that are not gravimetrically traceable to International System (SI)1 units and ( b ) as powders of unknown peptide content. In addition, for some peptides such as insulin and C-peptide, the most commonly used reference materials are no longer available. We recently developed an LC-MS/MS assay to simultaneously measure intact insulin and C-peptide (1). Here we describe rigorous characterization of peptide content of calibrators and controls, with the goal of mass-based standardization. We compare data from this approach to immunoassays that are standardized using the above-mentioned WHO reference materials.For our assay, we used human insulin WHO standard code 83/500 [National Institute for Biological Standards and Control (NIBSC)] and C-peptide (Anaspec) in the calibrators, and human insulin from US Pharmacopeia (USP) and C-peptide from Bachem in the QCs (1). Insulin, provided by NIBSC and USP as bulk powders, was weighed before dissolution (1). C-peptide from Anaspec and Bachem was provided as 0.5 mg of dried powder in glass vials. All solutions prepared from …