Quantitative Insulin Analysis Using Liquid Chromatography–Tandem Mass Spectrometry in a High-Throughput Clinical Laboratory

Abstract

BACKGROUND Circulating insulin concentrations reflect the amount of endogenous insulin produced by the pancreas and can be monitored to check for insulin resistance. Insulin is commonly measured using immunochemiluminometric assays (ICMA). Unfortunately, differing crossreactivities of the various ICMA antibodies have led to variability in assay results. In contrast, liquid chromatography–tandem mass spectrometry (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 resolution of the peptide was achieved by LC coupled to triple-quadrupole MS. Selected-reaction monitoring of B-chain transitions was used for quantification. Recombinant human 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 (13C/15N) 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 μIU/mL (10.8 pmol/L) and a limit of quantitation of 3 μIU/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 Deming 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 standard has been successfully developed and validated.

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry–Based Assays

“…When an assay reports concentrations on a population of samples that have very little bias when compared with a reference measurement procedure (i.e., a robust assay with rigorous process controls that uses certified or standard reference materials in its calibration), the assay is said to be standardized. It is possible to harmonize and standardize immunoassays or mass spectrometric assays (74). …”

Triglycerides and glucose index: A useful indicator of insulin resistance

“…On the one hand, insulin has a high biological variability (within-and between-subject variability of 21.1% and 58.3% respectively), 4 and on the other hand, its measurement has yet to be standardized. 5,6 These two aspects have a direct impact on the estimation of IR using the HOMA-IR index and other formulas which have been developed using insulin level in their calculations (QUICKI, FGIR, Raynaud, reciprocal insulin). 7 Because of these difficulties, attempts have been made to identify other parameters that could be helpful for assessing IR.…”

Development of a High-Sensitivity Quantitation Method for Arginine Vasopressin by High-Performance Liquid Chromatography Tandem Mass Spectrometry, and Comparison with Quantitative Values by Radioimmunoassay

“…The use of a 0.1% formic acid aqueous solution and acetonitrile improved the detection sensitivity of peptide quantitation. [23][24][25][26][27] However, in this work, our comparison revealed that methanol was better than acetonitrile in terms of sensitivity, and that a low acetic acid concentration (0.01%) was preferable over the use of formic acid. In general, acetonitrile is used in ESI ionization, because an organic solvent with low viscosity and a low boiling point is preferred for ionization.…”