1. 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 2013 www.clinchem.org/content/59/9/1349.full © Copyright 2013 by the American Association for Clinical Chemistry

2. © 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

3. © 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

4. © 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

5. © 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

6. © 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

7. © 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

8. © 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 243.6 pmol/L). RT: retention time; AA: peak area; SN: signal to noise ratio; BP: base peak RT:0.00 - 0.60 0.000.050.100.150.200.250.300.350.400.450.500.55 Time (min) 0 20 40 60 80 100 0 20 40 60 80 100 Relative Abundance 0 20 40 60 80 100 RT: 0.24 AA: 65415 SN:161 BP: 738.30 RT: 0.22 AA: 22211 SN:95 BP: 753.20 RT: 0.23 AA: 137231 SN:159 BP: 756.20 Bovine Insulin B Chain Human Insulin B Chain Human IS Insulin B Chain

9. © 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.

10. © 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:

11. © 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.

12. © 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 (7.1-14.0%)  Good low level sensitivity (LOQ: 3.0 µIU/mL)  Harmonization with NIBSC standard (66/304)  Only human endogenous insulin detected  No antibody used

13. © 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?

14. © Copyright 2009 by the American Association for Clinical Chemistry Thank you for participating in this month’s Clinical Chemistry Journal Club. Additional Journal Clubs are available at www.clinchem.org Download the free Clinical Chemistry app on iTunes for additional content! Follow us