Liquid Chromatography-Mass Spectrometry for the Determination of Antidepressants and Some of their Major Metabolites in Human Biological Matrices - LC-MS in Drug Bioanalysis - page 167

6.3.5 Relative Ions Intensity

Intraday imprecision of relative ions intensity for each analyte was evaluated by
analysis of five replicates at four concentration levels analyzed on the same day,
and interday imprecision on this parameter was calculated at the same concen-
tration levels ( n = 5 each) analyzed on five different days. %CV for the relative
ions intensities was <17% in all cases, except for norfluoxetine, for which %CV
was <26%.

6.3.6 Extraction Recovery

Extraction recovery was assessed at low and high concentrations by comparing
analyte-to-IS peak area ratio when standards were added before extraction ( n = 5) to
that obtained when added after extraction ( n = 5). IS mixture was added after extrac-
tion in all cases. Recoveries ranged from 49 to 72 % for all analytes.

6.3.7 Matrix Effect

Matrix effect was initially evaluated by postcolumn infusion of a mixture contain-
ing the analytes and the ISs (1 mg/mL, 10 mL/min) in “T” with the effluent of the
chromatographic system. Chromatograms after the injection of extracted blank
plasma ( n = 6) and oral fluid samples ( n = 6) from different sources were compared
with the chromatograms after the injection of mobile phase (no matrix effect).
Quantification of matrix effect was performed comparing average peak areas of
blank plasma ( n = 6) and oral fluid samples ( n = 6) fortified with the analytes
(100 ng/mL) and ISs after extraction to those obtained when the same amount of
analytes and ISs were added to a clean tube, evaporated and reconstituted in mobile
phase. Matrix effect was <15% for all analytes, except for norfluoxetine (signal
enhancement <45%) and paroxetine (signal enhancement <30%). Nevertheless,
inclusion of the deuterated analogues for these two analytes compensate for the
possible errors in imprecision, inaccuracy and recovery.

6.3.8 Stability Study

Antidepressant stability after three freeze-thaw cycles was evaluated in triplicate
at low and high concentrations. Calculated concentrations in the samples sub-
jected to these conditions (stability samples) were compared to those obtained in
freshly prepared samples (control samples). Stability and control samples were
quantified with a calibration curve prepared on the day of the analysis. All
analytes were stable under these conditions, except sertraline, for which a slight
signal decrease was observed at 250 ng/mL in oral fluid (MRE = −33.4%;
%CV = 6.0%).