Targetlynx quantification application manager software

For UPLC-MS/MS analysis, urine samples were
diluted with solvent A of UPLC gradient with isotopically labeled
internal standards and then applied on an UPLC-MS/MS instrument (ACQUITY
UPLC core system and a Quatro Premier XE triple quadrupole mass spectrometer
with an electrospray source, all from Waters Inc.). Samples were separated
on an Acquity UPLC HSS T3 (150 mm × 2.1 mm, 1.8 μm) column
(Waters Inc.) with a binary gradient (solvent A was 15 mM ammonia
acetate (pH 6.8) and solvent B was acetonitrile) at a flow rate of
0.45 mL/min. Optimized cone voltage and collision energy were used
for each individual analyte. For each analyte, three multiple reaction
monitoring (MRM) transitions were set up: one for quantification,
one for confirmation, and one for the labeled internal standard. These
MRMs were scheduled around the retention time of the analytes. Analytes
in urine samples were quantified using the peak area ratio based on
10-point standard curves, which were run before and after the urine
samples. The TargetLynx quantification application manager software
(Waters Inc.) was used for peak integration, calibration, and quantification.
The concentration values of analytes were normalized to the creatinine
level, which was measured on a COBAS MIRA-plus analyzer (Roche, NJ)
with Infinity Creatinine Reagent (Thermo Fisher Scientific).