Determination of free and protein-bound DA and NE, 5HT and Ach and their metabolites and oxidation products by UPLC-MS/MS method

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 four mixtures: MIX1 [dopamine (DA), noradrenaline (NA), 3-methoxytyramine (3MT), 3,4-dihydroxyphenylalanine (L-DOPA) and aminochrome (AC)], MIX2 [3,4-Dihydroxyphenylacetic

acid (DOPAC), 3,4-dihydroxymandelic acid (DOMA) and vanillylmandelic acid (VMA)], MIX3 [5SCD and 5SCDA] and MIX4 [serotonin(5-HT), tryptophan (Trp) and 5-hydroxyindole-3-acetic acid (5-HIAA)].

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

Serially dilute acetylcholine (ACh) standard with 0.1% volume to obtain a calibration curve covering a range of 0.2-1000 nM

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, MIX3 or MIX4), 96µL of 25millimolar (mM) and 24µL of 8micromolar (µM) .

For ACh calibration curve, distribute the sample into 90µL aliquots prior to the addition of 30µL of the working mixture and 120µL of 200nanomolar (nM) in 0.1% FA in ACN.

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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 55µL of sample for ACh determination: dilute 1:4 with 0.1% volume containing a final concentration of 100 nM of ACh-4d as IS.

Take 216µL for catecholaminergic and serotonergic determination and add 24µL of 8micromolar (µM) .

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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

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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

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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

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

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

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

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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).

An Acquity HSS T3 (1.8 μm, 2.1 mm × 100 mm) column coupled to an Acquity HSS T3 VanGuard (100 Å, 1.8 μm, 2.1 mm × 5 mm) pre-column is used to detect MIX1-3 analytes. Column temperature is set at 45 ºC and samples are maintained at 6 ºC in the thermostatic autosampler.

An Acquity UPLC BEH C18 (1.7μm, 2.1x100mm) column coupled to a Acquity BEH C18 1.7μM VanGuard pre-column is used to detect MIX4. Column temperature is set at 55 ºC and samples are maintained at 6ºC in the thermostatic autosampler.

A Cortecs UPLC HILIC (1.6 µm; 2,1x75 mm) column coupled to a Cortecs UPLC HILIC VanGuard pre-column (Waters) is used for ACh determination. Column temperature is set at 50 ºC and samples

are maintained at 6 ºC in the thermostatic autosampler.

The mobile phase for MIX1-4 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.

0.5

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 maintained 0.2 min for equilibration. Total run time 3.7 min

MIX4:

1%B maintained for 0.5 min, 25 % B at 3 min, 50 % B at 3.1 min and maintained for 0.5 min, 1 % B at 3.6 min maintained 0.4 min for equilibration.

The mobile phase for ACh determination consisted of solvent A (0.1% FA in ACN) and solvent B (10 mM ammonium acetate in MilliQ water) at a flow of 0.5 mL/min with isocratic 70% A- 30% B conditions during 2.2 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 was set at: 0.5 kV (MIX1, MIX3 and MIX3-PB), 2 kV (MIX2) or 3kV (MIX4, ACh). The electrospray ionization (ESI) source was operated in both positive and negative modes, depending on the analyte.

Multiple Reaction Monitoring (MRM) acquisition settings for the targeted metabolites are

summarised in the following Table

A	B	C	D	E	F	G
Table 1. MRM acquisition settings a
Analyte	MRM transition (m/z)	MIX	RT (min)	CV (V)	CE (eV)	CpV (kV)
ACh	145,98 > 86,80	ACh	1,5	10	15	3
ACh-d4	150 > 91	ACh	1,5	28	12	3
NE b	151,75 > 106,94	1	0,69	15	20	0,5
DA-d4 (IS)	157,83 > 94,8	1,2,3	1,44	10	20	0,5
DA	153,93 > 90,57	1	1,46	10	20	0,5
L-DOPA	198,1 > 152,1	1	1,48	15	15	0,5
3MT b	150,7 > 90,96	1	3,09	35	20	0,5
AC	149,61 > 121,91	1	3,36	25	25	0,5
DOMA c	182,86 > 136,85	2	1,62	20	14	2
VMA c	197 > 136,9	2	3,61	20	20	2
DOPAC c	166,99 > 122,82	2	3,72	18	22	2
5SCDA	273,1 > 166,9	3	1,73	20	20	0,5
5SCD	317 > 154,86	3	2,01	24	30	0,5
5HT	177 > 160	4	0,97	10	5	3
5HT-d4 (IS)	181 >164	4	0,97	10	5	3
5HIAA	192 > 146	4	1,5	25	20	3
Trp	205 > 188	4	2,1	15	10	3
a RT, retention time; D, dwell time; CV, cone voltage; CE, collision energy; CpV, capillary voltage.
b Parent mass after loss of water.
c Detected in negative mode.

Samples with a concentration between the limit of detection (LOD) and limit of quantification (LOQ) or bigger than LOQ are considered acceptable; samples with a concentration lower than LOD are considered

as the LOD value.

Catechol oxidation is measured using the formula AC+5SCDA+5SCDA-PB/DA + 5SCD+5SCD-PB/L-DOPA

DA synthesis is measured using the formula DA+NE+DOMA+VMA+ 3MT+DOPAC /L- DOPA

DA degradation is measured using the formula DOPAC+3MT/DA

Data is normalised by the protein concentration (determined by BCA) and presented as the percentage of the wt concentration or ratio.