quantitative insulin analysis using liquid chromatography–tandem mass spectrometry in a high-...
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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
© Copyright 2009 by the American Association for Clinical Chemistry
BackgroundBackground
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
BackgroundBackground
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
BackgroundBackground
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
BackgroundBackground
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
BackgroundBackground
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
MethodologyMethodology
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© Copyright 2009 by the American Association for Clinical Chemistry
Results: Insulin in Patient’s serumResults: 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
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55
Time (min)
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Rel
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ance
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RT: 0.24AA: 65415SN: 161BP: 738.30
RT: 0.22AA: 22211SN: 95BP: 753.20
RT: 0.23AA: 137231SN: 159BP: 756.20
Bovine Insulin B Chain
Human Insulin B Chain
Human IS Insulin B Chain
© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry
Results: Analytical PerformanceResults: Analytical Performance
Table 1. Performance of the LC-MS/MS assay for insulin. 150 µl of patient serum extracted per analysis.
Sensitivity LOB LOD LOQ 1.4 IU/mL 1.8 IU/mL 3.0 IU/mL (8.4 pmol/L) (10.8 pmol/L) (18.0 pmol/L) Precision Insulin in Stripped Inter-assay Intra-assay Serum, IU/mL %CV, % accuracy %CV, % accuracy
(n=8) (n=8) 8 14.0, 91.3 7.0, 80.0 12 10.2, 91.2 7.9, 92.2 20 10.0, 87.0 6.0, 86.5 40 7.5, 87.7 4.0, 87.6 80 7.1, 96.3 3.0, 92.1 Recovery Insulin in serum Mean % recovery IU/mL (n=3) 8 93.8 20 113.3 48 99.4
© Copyright 2009 by the American Association for Clinical Chemistry© 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:Results:
Scatter Plot with Deming Fit
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Insulin by Commercial ICMA (µIU/mL)
Ins
ulin
by
LC
MS
MS
(µIU
/mL
) Identity
Deming fit(-0.89 + 1.15x)
© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry
0
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/MS
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IU/m
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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
DiscussionDiscussion
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
© Copyright 2009 by the American Association for Clinical Chemistry
QuestionsQuestions
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?
© Copyright 2009 by the American Association for Clinical Chemistry
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