Quantitative Insulin Analysis Using Liquid Chromatography–Tandem Mass Spectrometry in a High- Throughput Clinical Laboratory Z. Chen, M.P. Caulfield, M.J. McPhaul, R.E. Reitz, S.W. Taylor, and N.J. Clarke September © Copyright 2013 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry Background  The measurement of fasting insulin concentrations is one method to assess patients for insulin resistance  Immunological techniques are currently widely used for the insulin detection  A mass spectrometry-based assay has been developed as an alternative for the routine measurement of insulin concentrations

© Copyright 2009 by the American Association for Clinical Chemistry Background  Immunoassay platforms have limitations  Results not standardized across platforms mainly due to antibody cross-reactivities  Auto- or heterophilic antibodies may introduce biases  May not differentiate native insulin from insulin analogs

© Copyright 2009 by the American Association for Clinical Chemistry Background  A clinically challenging assay for mass spectrometry  Low endogenous insulin concentration requires high analytical sensitivity  Matrix complexity requires high analytical specificity  High throughput & robustness required  Do not want to use antibodies in sample preparation

© Copyright 2009 by the American Association for Clinical Chemistry Background  Human Insulin  Molecular weight: 5808 Da  Two peptide chains (A & B) connected by two disulfide bonds  Reducing agent separates insulin A and B chains into individual peptides

© Copyright 2009 by the American Association for Clinical Chemistry Background  Advantages of detecting insulin B chain  Simple reduction liberates B Chain  B chain is more mass spec “friendly”  Decreases the matrix background  Simpler, faster and more robust than enzymatic digestion

© Copyright 2009 by the American Association for Clinical Chemistry Methodology  Procedure  150 µL patient serum mix with basic ethanol  Reducing agent added into supernatant  Inject sample onto LC-MS/MS  Two Dimensional LC-MS/MS system  Turbo-flow Aria TLX (Thermo Fisher)  Thermo Fisher TSQ Vantage triple quadrupole mass spectrometer

© Copyright 2009 by the American Association for Clinical Chemistry Results: Insulin in Patient’s serum Figure 1. Example of chromatograms of a patient’s serum (40.6 µIU/mL or pmol/L). RT: retention time; AA: peak area; SN: signal to noise ratio; BP: base peak RT: Time (min) Relative Abundance RT: 0.24 AA: SN:161 BP: RT: 0.22 AA: SN:95 BP: RT: 0.23 AA: SN:159 BP: Bovine Insulin B Chain Human Insulin B Chain Human IS Insulin B Chain

© Copyright 2009 by the American Association for Clinical Chemistry Results: Analytical Performance Table 1. Performance of the LC-MS/MS assay for insulin. 150 µl of patient serum extracted per analysis.

© Copyright 2009 by the American Association for Clinical Chemistry Figure 2. Method comparison (Deming Regression) LC-MS/MS vs. FDA-approved ICMA platform for 89 patient samples. Results:

© Copyright 2009 by the American Association for Clinical Chemistry Figure 3. A reference interval for insulin determined by LC-MS/MS for 97 healthy donors.

© Copyright 2009 by the American Association for Clinical Chemistry Discussion  Successful routine LC-MS/MS insulin assay  Rapid assay: 2 min/sample, <4h/96w-plate  High throughput and fully automatic  Good CVs ( %)  Good low level sensitivity (LOQ: 3.0 µIU/mL)  Harmonization with NIBSC standard (66/304)  Only human endogenous insulin detected  No antibody used

© Copyright 2009 by the American Association for Clinical Chemistry Questions  Why was immunocapture not a good choice to prepare the sample for this assay?  Is high resolution mass spectrometry an option for this assay?  What are the advantages and disadvantages of using an insulin assay for the assessment of pre-diabetes and diabetes compared to other measurements?

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