Determination of Antidepressants in Human Plasma by Modified Cloud-Point Extraction Coupled with Mass Spectrometry

Abstract

Cloud-point extraction (CPE) is rarely combined with liquid chromatography coupled to mass spectrometry (LC-MS) in drug determination due to the matrix effect (ME). However, we have recently shown that ME is not a limiting factor in CPE. Low extraction efficiency may be improved by salt addition, but none of the salts used in CPE are suitable for LC-MS. It is the first time that the influences of a volatile salt-ammonium acetate (AA)-on the CPE extraction efficiency and ME have been studied. Our modification of CPE included also the use of ethanol instead of acetonitrile to reduce the sample viscosity and make the method more environmentally friendly. We developed and validated CPE-LC-MS for the simultaneous determination of 21 antidepressants in plasma that can be useful for clinical and forensic toxicology. The selected parameters included Triton X-114 concentration (1.5 and 6%, w/v), concentration of AA (0, 10, 20 and 30%, w/v), and pH (3.5, 6.8 and 10.2). The addition of 10% of AA increased recovery twice. For 20 and 30% (w/v) of AA, three phases were formed that prolonged the extraction process. The developed CPE method (6% Triton X-114, 10% AA, pH 10.2) was successfully validated through LC-MS/MS simultaneous determination of 21 antidepressants in human plasma. The linearity was in the range of 10-750 ng/mL (r² > 0.990).

Keywords: LC–MS/MS; antidepressant; bioanalytical methods; sample preparation.

Figure A1

Multiple reaction monitoring (MRM) chromatogram peaks of the analyzed antidepressants extracted from (a) blank plasma; (b) quality control at a concentration of 625 ng/mL using cloud-point extraction. The MS parameters of MRM mode are presented in Table 3.

Figure A2

Recovery in the selected CPE method conditions (pH 10.2, 6% Triton X-114, 10% (w/v) ammonium acetate) determined for 6 different sources of plasma.

Figure A3

Variants of samples tested during CPE development.

Figure 1

Chemical structures of antidepressants studied in the experiment.

Figure 2

Effects of Triton X-114 concentration on (a) recovery (p < 0.001), (b) absolute matrix effect (ME_A) (p = 0.182). Extraction conditions: equilibrium temperature—60°C; equilibrium time—20 min; sample pH 6.8; ammonium acetate content—20% (w/v). Antidepressants level corresponds to a plasma concentration of 100 ng/mL Higher mean recovery was observed with higher Triton X-114 concentration (the experiment was repeated three times) (p < 0.001). Results are presented as means and standard deviations.

Figure 3

The effect of ammonium acetate concentration (0, 10, 20, and 30% (w/v)) on recovery (p < 0.001). Extraction conditions: equilibrium temperature—60 °C; equilibrium time—20 min; sample pH, 6.8; concentration of Triton X-114–6% (w/v). Antidepressants level corresponds to a plasma concentration of 100 ng/mL. Results are presented as means and standard deviations. Mean recoveries for 0% AA (dashed line), 10% AA (dotted line), 20% AA (dotted-dashed line), and 30% AA (bold dashed) are shown.The highest increases of recovery, as a result of AA addition, were observed for AMI, PAR, PRO, TIA, and TRA, whereas the weakest effect was recorded for both FLV and MIA. No increase in recovery for VEN or decrease for OPI was observed. The increase of recovery (10% vs. 0 % (w/v)) was correlated (r_s = 0.465, p = 0.034) with the calculated octanol–water partition coefficient (cLogP).

Figure 4

Effect of the addition of ammonium acetate on the absolute matrix effect (ME_A) (p = 0.198). Extraction conditions: equilibrium temperature—60°C; equilibrium time—20 min; sample pH—6.8; concentration of Triton X-114—6% (w/v). Antidepressants level corresponds to a plasma concentration of 100 ng/mL. Results are presented as means and standard deviations. ME_A between 85% (dotted line) and 115% (dotted line) was considered insignificant.

Figure 5

The effects of different pH of a sample on (a) recovery (p < 0.001), (b) absolute matrix effect (ME_A) (p = 0.514). Results are presented as means and standard deviations. ME_A between 85% (dashed line) and 115% (dashed line) was considered as insignificant. Extraction conditions: Triton X-114 at 6% (w/v); ammonium acetate at 10% (w/v); equilibrium temperature—60°C; equilibrium time—20 min. Antidepressants level corresponds to plasma concentration of 100 ng/mL.

Figure 6

The effects of cloud-point extraction conditions on (a) the recoveries and (b) the absolute matrix effects of 21 antidepressants determined using liquid chromatography coupled with mass spectrometry. T—Triton concentration (w/v), AA—ammonium acetate concentration (w/v).

Figure 7

Distribution of the analyzed compounds on a score plot (principal component 2 vs. component 1) in a principal component analysis (PCA). The results are presented for the optimal CPE variant (pH 10.2; 6% Triton X-114, 10% (w/v) ammonium acetate; equilibrium temperature 60°C; equilibrium time 20 min). HBA—number of hydrogen bond acceptors, HBD—number of hydrogen bond donors, PSA—polar surface area. Compounds labelled with green text exhibited insignificant ME_A, whereas compounds labeled with red—significant ME_A.