Quantitative Insulin Analysis Using Liquid Chromatography–Tandem Mass Spectrometry in a High-Throughput Clinical Laboratory Zhaohui Chen, 1 Michael P. Caulfield, 1 Michael J. McPhaul, 1 Richard E. Reitz, 1 Steven W. Taylor, 1 and Nigel J. Clarke 1* BACKGROUND: Circulating insulin concentrations re- flect the amount of endogenous insulin produced by the pancreas and can be monitored to check for insulin resistance. Insulin is commonly measured using im- munochemiluminometric assays (ICMA). Unfortu- nately, differing crossreactivities of the various ICMA antibodies have led to variability in assay results. In contrast, liquid chromatography–tandem mass spec- trometry (LC-MS/MS)-based approaches can provide a highly specific alternative to immunoassays. METHODS: Insulin was extracted from patient serum and reduced to liberate the insulin B chain. Subsequent reso- lution of the peptide was achieved by LC coupled to triple- quadrupole MS. Selected-reaction monitoring of B-chain transitions was used for quantification. Recombinant hu- man insulin was used as a calibrator and was compared against the National Institute for Biological Standards and Control (NIBSC) reference standard. Bovine insulin and a stable isotopic-labeled ( 13 C/ 15 N) human insulin B chain were used and compared as internal standards. RESULTS: The LC-MS/MS assay described herein has been validated according to CLIA guidelines with a limit of detection of 1.8 IU/mL (10.8 pmol/L) and a limit of quantitation of 3 IU/mL (18.0 pmol/L). A correlation between the LC-MS/MS assay and a US Food and Drug Administration-approved ICMA was completed for patient samples and the resulting Dem- ing regression revealed good agreement. A reference interval for the assay was established. CONCLUSIONS: A simple, high-throughput, quantitative LC-MS/MS insulin assay traceable to the NIBSC stan- dard has been successfully developed and validated. © 2013 American Association for Clinical Chemistry The detection of individuals with prediabetes and dia- betes may be achieved by measurement of fasting glu- cose, glycohemoglobin, and fasting insulin concentra- tions and glucose concentrations following an oral glucose load. Each of these methods is effective, but each has drawbacks. Fasting glucose concentrations that are not within reference intervals reflect a physio- logical state in which early diabetes has already devel- oped. Increases in hemoglobin A 1C can reflect either the latter stages of prediabetes or overt diabetes. Oral glucose tolerance testing may identify insulin resis- tance earlier, but it involves a more involved dynamic testing and may not always be amenable to the clinical testing environment. The measurement of fasting serum insulin has been suggested as potentially providing a rapid and readily accessible test to complement other existing methodologies. Indeed, numerous studies have dem- onstrated increased concentrations of fasting insulin in patients with prediabetes, even in the absence of in- creases of fasting glucose and hemoglobin A 1C (1–5 ). In a limited number of circumstances, the utility of fasting insulin measurements to detect early insulin re- sistance has been suggested (2, 6 ). Immunological techniques have been widely used for insulin quantification, initially through radioim- munoassay, and more recently by commercially avail- able immunochemiluminometric assays (ICMAs) 2 on automated platforms (7). However, no international reference method for insulin has yet been established. The major hurdle in establishing such a method stems from the variability in insulin values measured across different immunoassays and platforms. Measured val- ues can differ by a factor of 2 for the same WHO human insulin standard (7). The differences in results from various ICMA platforms are likely caused by differing cross-reactivities of the assay antibodies used. Further- 1 Quest Diagnostics Nichols Institute, San Juan Capistrano, CA. * Address correspondence to this author at: Quest Diagnostics Nichols Institute, 33068 Ortega Hwy, San Juan Capistrano, CA, 92675. Fax 949-728-4872; e-mail Nigel.J.Clarke@questdiagnostics.com. Received November 20, 2012; accepted April 26, 2013. Previously published online at DOI: 10.1373/clinchem.2012.199794 2 Nonstandard abbreviations: ICMA, immunochemiluminometric assays; MS, mass spectrometry; SRM, selected reaction monitoring; LC-MS/MS, liquid chromatography-tandem MS; TCEP, tris-(2-carboxyethyl) phosphine; NIBSC, Na- tional Institute for Biological Standards and Control; IS, internal standard; FDA, US Food and Drug Administration; SPE, solid-phase extraction; LOD, limit of detection; LOQ, limit of quantitation. Clinical Chemistry 59:9 1349–1356 (2013) Endocrinology and Metabolism 1349