Targeted Multiplexed Assay for Urinary Albumin Quantification Ashley Beasley-Green, Nijah M. Burris, David Bunk, Karen Phinney Biomolecular Measurement Division National Institute of Standards and Technology, Gaithersburg, MD ABSTRACT Urinary excretion of albumin is a major diagnostic and prognostic marker of renal dysfunction and cardiovascular disease; therefore, accurate measurement of urinary albumin is vital to clinical diagnosis. Although inter-method differences and analyte heterogeneity have been reported for urinary albumin measurements, accuracy assessments of the available methods have been hindered by the lack of a reference system, including reference measurement procedures and reference materials, for this clinical analyte. Therefore to address urinary albumin measurement precision, we have developed a reference measurement procedure that utilizes isotope dilution-mass spectrometry (ID-MS) and multiple reaction monitoring (MRM) to quantify full-length urinary albumin in a targeted mass spectrometric-based approach. The reference measurement procedure incorporates an isotopically- labeled (15N) full-length recombinant human serum albumin (15N-rHSA) material as the internal standard, which permits the absolute quantitation of albumin in urine. A total of 11 peptides with two transitions per peptide (22 MRM transitions) were selected from the tryptic digestion of human serum albumin on the basis of retention time reproducibility, peak intensity, and the degree of HSA sequence coverage. The repeatability and between-peptide comparability of each transition was evaluated to access the validity of the reference measurement procedure. In addition to method validation, the generated calibration curves were used to determine the albumin content in pooled human urine samples to access the accuracy of the MS-based urinary albumin quantitation method.