MSACL 2023 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.