Aug 09, 2024

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

DOI
dx.doi.org/10.17504/protocols.io.8epv5rmj6g1b/v1
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  • Vilalab Public
Miquel Vila
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DOI: dx.doi.org/10.17504/protocols.io.8epv5rmj6g1b/v1
Protocol Citation: Miquel Vila, Marta Gonzalez-Sepulveda 2024. Determination of free and protein-bound DA and NE, 5HT and Ach and their metabolites and oxidation products by UPLC-MS/MS method. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rmj6g1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 08, 2024
Last Modified: August 09, 2024
Protocol Integer ID: 104953
Abstract
Protocol for the determination of free and protein-bound DA and NE, 5HT and Ach and their metabolites and oxidation products by UPLC-MS/MS method
Materials
External standards

DA, DOPA, NE, DOPAC, DOMA, DOPE, VMA --> up to 3000 nM in 25 mM FA in water
3MT, AC, VMA, 5-SCDA, 5-SCD --> up to 1000 nM in 25 mM FA in water


Internal standard (IS)

DA-4d --> 500 nM in 25 mM FA in water






Preparation of the aminochrome (AC) external standard
Preparation of the aminochrome (AC) external standard
1m
1m
Mix Amount500 µL of Concentration1 millimolar (mM) dopamine (DA) with Amount500 µL of Concentration2 millimolar (mM) KIO4 disolved in Concentration100 micromolar (µM) aqueous ammonium acetate buffer Ph5.8 at RT with vigorous shaking for 1 min.

Note
Following oxidation, aminochromes are placed on ice to prevent further decomposition. Significant degradation of all aminochromes occurs at both RT and 4 ◦C within 24 h and -20ºC at 24-48h (Ochs 2005; Lemos-Amado 2001).

Preparation of calibration curves
Preparation of calibration curves
1h
1h
Prepare a stock solution of the IS in Concentration25 millimolar (mM) FA in water and store it at −80 °C.

Prepare fresh solutions of each metabolite in Concentration25 millimolar (mM) FA in water 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 Concentration25 millimolar (mM) FA in water to obtain the concentration series used in calibration curves.


Note
Tipically, final calibration levels cover a range of 1.72−3000 nM for DA, NE, and MIX2 and 0.39−1000 nM for L-DOPA, 3MT, AC and MIX3.


Serially dilute acetylcholine (ACh) standard with Concentration0.1 % volume FA in acetonitrile (ACN) 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 Concentration250 millimolar (mM) FA

Distribute the sample into Amount90 µL aliquots prior to the addition of Amount30 µL of the appropriate working mixture (MIX1, MIX2, MIX3 or MIX4), Amount96 µL of Concentration25 millimolar (mM) FA and Amount24 µL of Concentration8 micromolar (µM) IS (DA-4d + 5HT-4d) .


For ACh calibration curve, distribute the sample into Amount90 µL aliquots prior to the addition of Amount30 µL of the working mixture and Amount120 µL of Concentration200 nanomolar (nM) IS (ACh-4d) in 0.1% FA in ACN.
Centrifigation20000 rcf, 4°C, 00:10:00

10m
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 Amount7 µL into the UPLC-MS/MS system.
Sample preparation
Sample preparation
2h
2h
Add Amount300 µL of Concentration250 millimolar (mM) FA to each brain, intestine, heart or cell pellet sample prior homogenization. Dilute blood serum samples 1:10

Note
Due to the poor stability of aminochrome, usually a maximum of 50 samples can be analyzed at a time

Take a Amount20 µL 20 µl sample for protein determination (diluted 1/5 in Concentration25 millimolar (mM) FA )

Take Amount55 µL of sample for ACh determination: dilute 1:4 with Concentration0.1 % volume FA in ACN containing a final concentration of 100 nM of ACh-4d as IS.

Take Amount216 µL for catecholaminergic and serotonergic determination and add Amount24 µL of Concentration8 micromolar (µM) IS (DA-4d + 5HT-4d) .
Note
Important!!: ensure the concentration of IS is exactly the same in both calibration curves and samples


Centrifigation20000 rcf, 4°C, 00:10:00
Note
The supernatant is used to determine free neurotransmitters and metabolites (that is, those present in the deproteinated supernatant) while the pellet is used for protein-bound determinations (that is, those present in the acid-Insoluble pellet and released by HCl hydrolysis)

10m
Transfer supernatant to an Ostro protein precipitation and phospholipid removal plate (Waters, USA) to be filtered.
Inject Amount7 µL of filtered supernatant samples into the UPLC-MS/MS system
Reductive HCl hydrolysis of resulting pellets
Reductive HCl hydrolysis of resulting pellets
18h
18h

Safety information
Work in fume hood during all the procedure

After removal of the supernatant, wash the pellet (from both calibration curves and samples) with Amount1 mL of chloroform: methanol (1: 1, v/v) by vortex mixing

Centrifigation20000 rcf, 4°C, 00:10:00
10m
Transfer the resulting pellets to a sealed-capped tube with Concentration6 Molarity (M) HCl containing Concentration5 % volume thioglycolic acid and Concentration1 Mass Percent phenol
Note
- Calibration curves --> add Amount280 µL of the mixture and Amount40 µL of the corresponding calibration curve working mixture

- Samples --> add Amount288 µL of the mixture and Amount32 µL of IS



Purge tubes with a stream of nitrogen, seal them and heat them at Temperature110 °C forDuration16:00:00

16h
Let tubes cool at Temperature4 °C for at least Duration00:30:00

30m
Centrifigation20000 rcf, 4°C, 00:10:00
10m
Treat the supernatant with with acid-washed alumina to extract catecholic compounds
Alumina extraction of catecholic compounds
Alumina extraction of catecholic compounds
1h
1h
Transfer a Amount100 µL aliquot of each hydrolysate into a new Eppendorf tube containing Amount50 mg of acid-washed alumina and Amount200 µL of Concentration1 Mass Percent Na2S2O5 - Concentration1 Mass Percent EDTA.2Na

Add Amount500 µL of Concentration2.7 Molarity (M) Tris. HCl - Concentration2 Mass Percent EDTA.2Na
Ph9 to the mixture

Shaker1100 rpm, 22°C, 00:05:00 on a microtube mixer

5m
Centrifigation20000 rcf, 00:10:00
10m
Remove the aqueous layer by aspiration and wash alumina with Amount1 mL of Milli-Q water

Centrifigation20000 rcf, 00:10:00
10m
Remove the aqueous layer by aspiration and wash alumina with Amount1 mL of Milli-Q water

Centrifigation20000 rcf, 00:10:00
10m
Remove the aqueous layer by aspiration and wash alumina with Amount1 mL of Milli-Q water

Centrifigation20000 rcf, 00:10:00
10m
Remove the aqueous layer by aspiration
Elute catechols from alumina with Amount100 µL of Concentration0.4 Molarity (M) HClO4 by shaking for 2 min

2m
Collect all liquid into the injection plate without taking alumina
Note
Alumina is discarded after extraction

Finally, inject Amount7 µL into the UPLC-MS/MS system.
UPLC-MS/MS analysis for catecholaminergic, serotonergic and cholinergic determination
UPLC-MS/MS analysis for catecholaminergic, serotonergic and cholinergic determination
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
ABCDEFG
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.
Data analysis and representation
Data analysis and representation
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.