Self-Classified Topic Area(s): Troubleshooting > Assays Leveraging MS
Insufficient T3 Sensitivity for DBS Testing on SCIEX 6500 MS
Julian Reed Molecular Testing Labs
Julian Reed (Presenter) Molecular Testing Labs
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Free T3 is present in human serum at a level of 1.3-4.5 pg/mL. Due to the inherent sample volume limitations in dried blood spot testing, developing an assay with a limit of detection low enough for clinical relevance requires high sensitivity (0.5 pg/mL). Several methods were used to improve sensitivity: manual tuning, source parameter optimization, LC flow rate reduction, derivatization, column lipid cleanup, and use of a next-generation SCIEX 7500 instrument. Although quantifiable peaks were obtained in solvent standards, the sensitivity gains were not large enough to overcome matrix effects.
1. Problem: High sensitivity is needed to quantify free T3 in dried blood spots.
2. Method information:
a. Instrument: Shimadzu LC with MPX, SCIEX 6500 MS/MS
b. MP A: 0.1% Acetic Acid in Water
c. MP B: 0.1% Acetic Acid in ACN
d. Gradient: 2.5 minute gradient from 20% to 100% B at 0.4 mL/min
e. Column: Phenomenex Synergi 4 um Hydro RP 80 Å, 100 x 4.6 mm
f. MRM transitions: 651.7/197.0
3. Troubleshooting steps:
a.Source parameter tuning:
i. Gas Tune
ii. Temperature tune
iii. IonSpray Voltage tune
b.MRM mass tuning: Parent and daughter ion masses optimized via incremental mass adjustments of 0.1 amu.
c.MS/MS/MS: Attempted to reduce noise using QTRAP MS/MS/MS function, no gain in sensitivity.
d.Flow rate optimization: Flow rate varied to maximize signal
e.Derivatization: Butyl ester formation allows higher retention, greater sensitivity
f.Phospholipid transition: Several phospholipid transitions monitored for interference with T3 ionization.
4.Outcome:
a.Sensitivity of 0.5 pg/mL standard: S/N 13.7
b.This was slightly higher than the S/N obtained in SCIEX 7500 demo
c.Although neat solvent allowed quantitation at necessary levels, matrix effects in DBS samples swamped signal.
= Discovery stage. (24.37%, 2023)
= Translation stage. (39.50%, 2023)
= Clinically available. (36.13%, 2023)
