1290 liquid chromatography

An Agilent 1290 Liquid Chromatography (LC) system equipped with an autosampler and coupled to AB Sciex QTRAP 6500 mass spectrometer (MS) was used to quantify the corresponding monoamine and metabolites. Chromatographic separation was achieved on a Discovery HS F5 (150 mm x 4 mm, 3 µm, Sigma-Aldrich, St. Louis, MO, USA) pentafluorophenyl column thermostated at 37 o C. The mobile phase was water (A) and methanol (B) with 0.1% of HCOOH in both solvents. An increasing linear gradient (v/v) of B was used (t(min), %B), as follows, (0, 0), (0.5, 0), (5.90, 30), (6, 100), (9, 100), (9.10, 0), (10.0, 0) at a constant flow rate (500 µL/min). The flow was directed to waste for the first 2 min to prevent the inorganic ions of aCSF solution to A negative ion mode was used in the analysis of HVA (m/z 181 → 122).
Six standards (from 0.1 nM to 10 nM for DA or from 10 nM to 1 µM for metabolites)
were prepared daily in a solution composed by aCSF/antioxidant mixture (2:1) to obtain the calibration curve. The method showed linearity within the concentration range studied and the detection limit (signal-to-noise ratio=3) for DA was 0.05 nM and for DOPAC and HVA was 1 nM. The accuracy of the assay was 85 -115% and the intraand inter-assay coefficients of variation were less than 15%. Analyst v1.4.2 software was used to calculate the areas of chromatographic peaks.

An Agilent 1290 Liquid Chromatography (LC) system equipped with an autosampler and coupled to AB Sciex QTRAP 6500 mass spectrometer (MS) was used to quantify DA, 5-HT and metabolites. Chromatographic separation was achieved in a Discovery HS F5 (150 mm x 4 mm, 3 µm, Sigma-Aldrich, St. Louis, MO, USA) pentafluorophenyl column thermostated at 37 o C. The mobile phase was water (A) and methanol (B) with 0.1% of formic acid in both solvents. An increasing linear gradient (v/v) of B was used (t(min), %B), as follows, (0, 0), (0.5, 0), (5.90, 30), (6, 100), (9, 100), (9.10, 0), (10.0, 0) at a constant flow rate (500 µl/min). The flow was directed to waste for the first 2 min to prevent the inorganic ions of aCSF solution to enter the mass spectrometer. The microdialysate samples were refrigerated at 4 ºC and 20 µL was injected, without sample pretreatment, into the LC-MS/MS system. Mass spectrometric quantification in positive ion mode was carried out using the following transitions: DA (m/z 154 → 137 and 154 → 91, collision energies (CE) of 15 and 31 V, respectively), DOPAC (m/z 123 → 77, CE of 24 V), 5-HT (m/z 177 → 160, CE of 13 V) and 5-HIAA (m/z 192 → 146, CE of 23 V). A negative ion mode was used in the analysis of HVA (m/z 181 → 122, CE of -20 V).