Biomedical Engineering Reference
In-Depth Information
Fig. 3 Solid-phase extraction
(SPE) procedure applied to
plasma and oral fluid samples
for LC-MS/MS determination
of nine antidepressants and
some metabolites
SPE cartridge
OASIS MCX cartridge
2 mL MeOH
2 mL water
Conditioning
0.2 mL sample + 1 mL sodium
acetate buffer pH 3.6 + 50
Sample Load
L ISs
µ
mixture
2 mL formic acid 2% in water
2 mL MeOH
Clean up
positive pressure,
( N 2 flow, 5 min)
Dry cartridge
2 mL dichloromethane/2-
propanol/ammonium hydroxide
(75:24.5:0.5)
Elution
Evaporation (dryness)
35°C under a stream of N 2
0.1 mL (oral fluid) or 0.2 mL (plasma)
ammonium formate buffer
(pH 3.6, 2 mM)/ACN (85:15)
Reconstitution
Injection
20
µ
L LC-MS/MS
6.3.3
Limit of Detection (LOD) and Limit of Quanti fi cation (LLOQ)
LOD was defined as the lowest concentration for which the two monitored transi-
tions could be detected, and the peak area of the quantifier transition was, at least,
three times the background noise. LOD was 0.5 ng/mL for all analytes. LLOQ was
defined as the lowest concentration that could be quantified with appropriate
imprecision (coefficient of variation (%CV) <20%) and inaccuracy (mean relative
error (MRE) ± 20%). LLOQ was 2 ng/mL in oral fluid, and 2-4 ng/mL in plasma for
most of the analytes. Carryover after automated SPE of clomipramine at 1,000 ng/
mL was half of the signal obtained at 2 ng/mL; therefore, LLOQ in plasma was
increased to 10 ng/mL for this analyte.
6.3.4
Intra- and Interday Imprecision and Inaccuracy
Intraday imprecision and inaccuracy were evaluated at low, medium and high
concentrations within the validated concentration ranges for each matrix by analysis
of five replicates on the same day. Interday imprecision and inaccuracy were
assessed by analysis of five replicates at the same concentrations on five different
days. MRE was ±15% and %CV <17% in all cases.
 
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