Fig. 1. Scheme of on-line SPE system-LC (Symbiosis Pharma) coupled to the MS/MS. (A) The sample is loaded into the sample loop while the first SPE cartridge is conditioned in the left clamp. (B) The sample is loaded onto the cartridge followed by a washing step. (C) The cartridge is moved to the right clamp, where the sample is eluted; the analytes are subsequently retained on the focussing column. Simultaneously, a second sample is subjected to the initial procedure of extraction in the left clamp. (D) After the elution step the MS valve is switched and the analytes are now directed to the analytical column in connection to the MS/MS. Following the analysis of the first sample, the second SPE cartridge is transferred to the right clamp in order to be eluted and analysed.

Fig. 2. MRM chromatograms obtained following the analysis of a spiked plasma sample with 10 μg/L of (1) clomipramine, (2) amitriptyline, (3) sertraline, (4) imipramine, (5) norclomipramine, (6) nortriptyliline, (7) desipramine, (8) fluoxetine, (9) venlafaxine, (10) citalopram, (11) paroxetine, (12) norfluoxetine, (13) fluvoxamine and (14) trazodone. Peak intensity is shown on the right-hand corner of each trace.

Fig. 3. Post-column infusion experiments: matrix effect. (1) trazodone, (2) paroxetine, (3) nortriptyline and (4) amitriptyline, of an injection of a mobile phase control (A) and a blank sample following the extraction of plasma (B). The shaded area indicates the elution position of the respective antidepressants.

Fig. 4. Typical MRM chromatograms obtained following the analysis of one authentic plasma sample. Concentrations were 127 μg/L for venlafaxine (A) 1036 μg/L for trazodone (B). The figure shows the response for the two transitions of both compounds (quantifier and qualifier). Peak intensity is shown in the top right-hand corner of each trace.

MRM transitions and conditions for all the compounds and their deuterated analogues

Underlined transitions were used for quantification.

Reported therapeutic range [16] and linearity data for 14 antidepressants and their metabolites

Intra-assay and inter-assay precision and bias of the QC samples prepared in plasma at a concentration of 40, 200 and 800 μg/L

Extraction recovery and matrix effect

Data represent the mean of three experiments with a 1000 μg/L calibrator.

Comparative results when analysing the real samples with the on-line SPE–LC–MS/MS method and LC–DAD