Sep 13, 2022
Version 1
Determination of free and protein-bound DA and NE and their metabolites and oxidation products by UPLC-MS/MS method V.1
- 1Vall d'Hebron Research Institute
Protocol Citation: Miquel Vila, Miquel Vila 2022. Determination of free and protein-bound DA and NE and their metabolites and oxidation products by UPLC-MS/MS method. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6948rlqe/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: September 13, 2022
Last Modified: September 13, 2022
Protocol Integer ID: 69924
Abstract
Protocol for the determination of free and protein-bound DA and NE 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 500 µL of 1 millimolar (mM) dopamine (DA) with 500 µL of 2 millimolar (mM) KIO4 disolved in 100 micromolar (µM) aqueous ammonium acetate buffer 5.8 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 25 millimolar (mM) FA and store it at −80 °C.
Prepare fresh solutions of each metabolite in 25 millimolar (mM) FA 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 25 millimolar (mM) FA 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.
Homogenize control samples (i.e brain, intestines, heart, blood serum, cells...) in the appropriate volume of 250 millimolar (mM) FA
Distribute the sample into 90 µL aliquots prior to the addition of 30 µL of the appropriate working mixture (MIX1, MIX2 or MIX3), 96 µL of 25 millimolar (mM) FA and 24 µL of 8 micromolar (µM) IS .
20000 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 7 µL into the UPLC-MS/MS system.
Sample preparation
Sample preparation
2h
2h
Add 300 µL of 250 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 20 µL 20 µl sample for protein determination (diluted 1/5 in 25 millimolar (mM) FA )
Take 240 µL for metabolite determination and add 26 µL of IS
Note
Important!!: ensure the concentration of IS is exactly the same in both calibration curves and samples
20000 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 7 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 1 mL of chloroform: methanol (1: 1, v/v) by vortex mixing
20000 rcf, 4°C, 00:10:00
10m
Transfer the resulting pellets to a sealed-capped tube with 6 Molarity (M) HCl containing 5 % volume thioglycolic acid and 1 Mass Percent phenol
Note
- Calibration curves --> add 280 µL of the mixture and 40 µL of the corresponding calibration curve working mixture
- Samples --> add 288 µL of the mixture and 32 µL of IS
Purge tubes with a stream of nitrogen, seal them and heat them at 110 °C for16:00:00
16h
Let tubes cool at 4 °C for at least 00:30:00
30m
20000 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 100 µL aliquot of each hydrolysate into a new Eppendorf tube containing 50 mg of acid-washed alumina and 200 µL of 1 Mass Percent Na2S2O5 - 1 Mass Percent EDTA.2Na
Add 500 µL of 2.7 Molarity (M) Tris. HCl - 2 Mass Percent EDTA.2Na
9 to the mixture
1100 rpm, 22°C, 00:05:00 on a microtube mixer
5m
20000 rcf, 00:10:00
10m
Remove the aqueous layer by aspiration and was alumina with 1 mL of Milli-Q water
20000 rcf, 00:10:00
10m
Remove the aqueous layer by aspiration and was alumina with 1 mL of Milli-Q water
20000 rcf, 00:10:00
10m
Remove the aqueous layer by aspiration and was alumina with 1 mL of Milli-Q water
20000 rcf, 00:10:00
10m
Remove the aqueous layer by aspiration
Elute catechols from alumina with 100 µL of 0.4 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 7 µL into the UPLC-MS/MS system.
UPLC-MS/MS analysis
UPLC-MS/MS analysis
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 mobile 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.