Mix 500µL of 1millimolar (mM) with 500µLof 2millimolar (mM) disolved in 100micromolar (µM) aqueous ammonium acetate buffer 5.8 at RT with vigorous shaking for 1 min.

Prepare a stock solution of the IS in 25millimolar (mM) and store it at −80 °C.

Prepare fresh solutions of each metabolite in 25millimolar (mM) and use them to make three mixtures: MIX1 (DA, L-DOPA, NE, 3MT, AC), MIX2 (DOMA, DOPE, DOPAC) and MIX3 (5SCD and 5SCDA).

Serially dilute mixtures with 25millimolar (mM) to obtain the concentration series used in calibration curves.

Homogenize control samples (i.e brain, intestines, heart, blood serum, cells...) in the appropriate volume of 250millimolar (mM)

Distribute the sample into 90µL aliquots prior to the addition of 30µL of the appropriate working mixture (MIX1, MIX2 or MIX3), 96µL of 25millimolar (mM) and 24µL of 8micromolar (µM) .

20000rcf,4°C

Transfer supernatant to an Ostro protein precipitation and phospholipid removal plate (Waters, USA) to filter it.

Save the pellet for protein-bound determinations (see below)

Finally, inject 7µL into the UPLC-MS/MS system.

Add 300µL of 250millimolar (mM) to each brain, intestine, heart or cell pellet sample prior homogenization. Dilute blood serum samples 1:10

Take a 20µL 20 µl sample for protein determination (diluted 1/5 in 25millimolar (mM) )

Take 240µL for metabolite determination and add 26µL of IS

20000rcf,4°C

Transfer supernatant to an Ostro protein precipitation and phospholipid removal plate (Waters, USA) to be filtered.

Inject 7µL of filtered supernatant samples into the UPLC-MS/MS system

After removal of the supernatant, wash the pellet (from both calibration curves and samples) with 1mLof chloroform: methanol (1: 1, v/v) by vortex mixing

20000rcf,4°C

Transfer the resulting pellets to a sealed-capped tube with 6Molarity (M) containing 5% volume and 1Mass Percent

Purge tubes with a stream of nitrogen, seal them and heat them at 110°C for16h 0m 0s

Let tubes cool at 4°C for at least 0h 30m 0s

20000rcf,4°C

Treat the supernatant with with acid-washed alumina to extract catecholic compounds

Transfer a 100µL aliquot of each hydrolysate into a new Eppendorf tube containing 50mg of acid-washed alumina and 200µL of 1Mass Percent - 1Mass Percent

Add 500µL of 2.7Molarity (M) - 2Mass Percent

9 to the mixture

1100rpm

20000rcf

Remove the aqueous layer by aspiration and was alumina with 1mL of Milli-Q water

20000rcf

Remove the aqueous layer by aspiration and was alumina with 1mL of Milli-Q water

20000rcf

Remove the aqueous layer by aspiration and was alumina with 1mL of Milli-Q water

20000rcf

Remove the aqueous layer by aspiration

Elute catechols from alumina with 100µL of 0.4Molarity (M) by shaking for 2 min

Collect all liquid into the injection plate without taking alumina

Finally, inject 7µL into the UPLC-MS/MS system.

A Waters Acquity™ UPLC system is coupled with a Xevo TQ-S triple quadrupole mass spectrometer with electrospray ionization interface (Waters). Instrument control, data acquisition, and analysis is performed using MassLynx V4.1 (Waters).

Chromatographic separation of samples is performed on a Waters Acquity™ HSS T3 (1.8μm; 2.1x100mm) column coupled to an Acquity™ HSS T3 VanGuard (100Å, 1.8 µm, 2.1 mm X 5 mm) pre-column (Waters). Column temperature is set at 45 ºC and samples are maintained at 6 ºC in the thermostatic autosampler.

The phase consisted of solvent A (methanol 100%) and solvent B (25 mM FA in MQ water) at a flow of 0.4 mL/min with the following gradient profiles (depending on the MIX):

MIX1 and MIX2:

0.5% B maintained for 0.5 min, 5% B at 0.9 min and maintained for 2.1min, 50% B at 2.8 min and maintained for 1.2 min, 0.5% B at 4.1 min followed by 0.2 min of equilibration. Total run time 4.3 min.

MIX3:

0.5% B maintained for 0.5 min, 8% B at 2.6 min, 50% B at 2.9 min and maintained for 0.6 min, 0.5% B at 3.7 min. Total run time 3.7 min

The mass spectrometer detector operates under the following parameters: source temperature 150 ºC, desolvation temperature 450 ºC, cone gas flow 50 L/hr, desolvation gas flow 1100 L/hr and collision gas flow 0.15 mL/min. Argon is used as the collision gas. The capillary voltage is set at 0.5 kV for MIX1 and MIX3, and at 2 kV for MIX2 detection. The electrospray ionization (ESI) source was operated in both positive and negative modes, depending on the analyte.