Biomedical Engineering Reference
In-Depth Information
matographic separation at 26 °C. Mobile phase was ammonium formate buffer
(pH 3.0; 2 mM) and ACN at a flow rate of 0.4 mL/min, applying the following
gradient: 15% ACN for 0.5 min, increased to 50% over 3.5 min, and increased again
to 70% at minute 5. With these conditions, all compounds eluted within 5 min, with
a total run time of 8 min. Figure
2
shows the chromatograms of the 13 antidepres-
sants in oral fluid at the LLOQ (2 ng/mL).
For the detection, a tandem mass spectrometer Quattro Micro™ API ESCI
(Waters Corp., Milford, MA) with a triple quadrupole was employed. The instru-
ment was operated in electrospray in the positive ionization mode (ESI+) with the
following optimized parameters: capillary voltage, 0.5 kV; source block tempera-
ture, 130 °C; nebulization and desolvation gas (nitrogen) heated at 400 °C and
delivered at 800 L/h, and as cone gas at 50 L/h; collision cell pressure, 3 × 10
-6
bar
(argon). Data was recorded in the multiple reaction monitoring (MRM) mode by
selection of the two most intense precursor-to-product ion transitions for each ana-
lyte, except for the ISs, for which only one transition was monitored. The most
intense transition for each analyte was used for quantitative purposes. Table
2
shows
MRM transitions, cone voltages and collision energies used for the analysis of the
antidepressants included in the LC-MS/MS method.
6.2
Oral Fluid and Plasma Extraction Procedure
Solid phase extraction (SPE) was performed with an ASPEC XL automated system
(Gilson, Middletown, USA) and mixed mode OASIS MCX cartridges 60 mg 3 cm
3
(Waters Corp., Milford, USA). Before SPE, 1 mL of sodium acetate buffer pH 3.6
and 50 mL of IS mixture (nortriptyline-d3, clomipramine-d3, paroxetine-d6,
norfluoxetine-d6, and fluoxetine-d6) at 0.2 mg/L in oral fluid and 0.4 mg/L in
plasma were added to 0.2 mL of sample. The applied SPE procedure is summa-
rized in Fig.
3
.
6.3
Method Validation
LC-MS/MS method validation was performed as follows:
6.3.1
Selectivity
Selectivity
of the method was evaluated by analysis of blank oral fluid and plasma
specimens from ten healthy subjects. In addition, potential exogenous interferences
were assessed by analysis of authentic plasma specimens containing other common
therapeutic drugs like benzodiazepines and/or drugs of abuse. No endogenous or
exogenous interferences were found in the monitored MRM channels in any of the
analyzed specimens.
