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2 Instrument Parameters
A
powered by TurboFlow technology was used to run the
LC-MS/MS method. The TurboFlow online extraction
column was a Thermo Scientific™ TurboFlow™ C18XL
column (50 x 0.5 mm) and the analytical column was a
1.9 µm
50 x 2.1 mm). Chromatography gradient information is
provided in Table 1 for two-dimensional TurboFlow
chromatography.
A
as operated in positive
ion mode with an atmospheric pressure chemical
ionization (APCI) source. The details are provided in
Table 2. Selected-reaction monitoring (SRM) transitions
are listed in Table 3. Data were acquired using
ith
Thermo Scientific ™Aria™ system control. Data analysis
was performed usin
Results and Discussion
Measurement of MPA by LC-MS/MS is not without its
challenges, as it is possible to observe interferences in
samples that are not observed in commercial or blank
plasma spiked with MPA. For example, interference
from the ac0079l glucuronide metabolite of MPA,
MPAG, should be considered since it breaks down in
the ionization source to MPA. This occurs in both
electrospray ionization (ESI) and APCI. Therefore, it
is essential to ensure that MPA and MPAG are
chromatographically separated.
Table 1. HPLC gradient table for 2D TurboFlow chromatography
Loading Pump Eluting Pump
Step Start Sec Flow
mL/
min
Gradient %A %B %C %D Tee Loop Flow
mL/
min
Gradient %A %B %C %D
1 00:00 40 2.00 Step 100 - - - - Out 0.60 Step 90 10 - -
2 00:40 80 0.20 Step 100 - - - T In 0.40 Step 90 10 - -
3 02:00 120 2.00 Step - - 100 - - In 0.60 Ramp - 100 - -
4 04:00 20 2.00 Step 10 90 - - - In 0.60 Step - 100 - -
5 04:20 60 2.00 Step 100 - - - - Out 0.60 Step 90 10 - -
Table 2. Source parameters for the MPA analysis in APCI mode
Parameter Value
Discharge current 6.0 µA
Sheath gas 50 arbitrary units
Aux gas 5 arbitrary units
Vaporizer temperature 400 °C
Capillary temperature 275 °C
Table 3. SRM transitions for MPA, MPAG, and internal standard (indomethacin)
Precursor Ion m/z Product Ion m/z Collision Energy Tube Lens
MPA 321.1 159, 207, 303 35, 23, 12 103
MPAG 514.0 321 18 103
Internal standard 357.9 111, 129, 138 43, 33, 22 108