TITLE: Determination of free and protein-bound DA and NE, 5HT and Ach and their metabolites and oxidation products by UPLC-MS/MS method
AUTHORS: Miquel Vila, Marta Gonzalez-Sepulveda

[DESCRIPTION]
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

[STEPS]
SECTION: Preparation of the aminochrome (AC) external standard
1. Mix 500 µL  of 1 millimolar (mM) with 500 µLof 2 millimolar (mM)  disolved in 100 micromolar (µM) aqueous ammonium acetate buffer pH 5.8 at RT with vigorous shaking for 1 min. 

 

SECTION: Preparation of calibration curves
2. Prepare a stock solution of the IS in 25 millimolar (mM) and store it at −80 °C.

Prepare fresh solutions of each metabolite in 25 millimolar (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)].
SECTION: Preparation of calibration curves
3. Serially dilute mixtures with  25 millimolar (mM) to obtain the concentration series used in calibration curves.


 


SECTION: Preparation of calibration curves
4. Homogenize control samples (i.e brain, intestines, heart, blood serum, cells...) in the appropriate volume of 250 millimolar (mM) 

SECTION: Preparation of calibration curves
5. Distribute the sample into 90 µL aliquots prior to the addition of 30 µL of the appropriate working mixture (MIX1, MIX2, MIX3 or MIX4), 96 µL of    25 millimolar (mM) and 24 µL of 8 micromolar (µM) .


SECTION: Preparation of calibration curves
6. 20000 rcf, 10 min, 4 °C 

SECTION: Preparation of calibration curves
7. 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)
SECTION: Preparation of calibration curves
8. Finally, inject 7 µL  into the UPLC-MS/MS system.
SECTION: Sample preparation
9. Add 300 µL  of 250 millimolar (mM) to each brain, intestine, heart or cell pellet sample prior homogenization. Dilute blood serum samples 1:10

 

SECTION: Sample preparation
10. Take a 20 µL 20 µl sample for protein determination (diluted 1/5 in  25 millimolar (mM) ) 

SECTION: Sample preparation
12. Take 216 µL for catecholaminergic and serotonergic determination and add 24 µL  of 8 micromolar (µM) . 
 
 


SECTION: Sample preparation
13. 20000 rcf, 10 min, 4 °C 
 

SECTION: Sample preparation
14. Transfer supernatant to an Ostro protein precipitation and phospholipid removal plate (Waters, USA) to be filtered.
SECTION: Reductive HCl hydrolysis of resulting pellets
16. 
 

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

SECTION: Sample preparation
15. Inject  7 µL of filtered supernatant samples into the UPLC-MS/MS system
SECTION: Reductive HCl hydrolysis of resulting pellets
17. 20000 rcf, 10 min, 4 °C 
SECTION: Reductive HCl hydrolysis of resulting pellets
18. Transfer the resulting pellets to a sealed-capped tube with 6 Molarity (M) containing 5 % volume and 1 Mass Percent 
 


SECTION: Reductive HCl hydrolysis of resulting pellets
19. Purge tubes with a stream of nitrogen, seal them and heat them at 110 °C for960 min 

SECTION: Reductive HCl hydrolysis of resulting pellets
20. Let tubes cool at 4 °C for at least 30 min 

SECTION: Reductive HCl hydrolysis of resulting pellets
21. 20000 rcf, 10 min, 4 °C 
SECTION: Reductive HCl hydrolysis of resulting pellets
22. Treat the supernatant with with acid-washed alumina to extract catecholic compounds
SECTION: Alumina extraction of catecholic compounds
23. 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 - 1 Mass Percent   

SECTION: Alumina extraction of catecholic compounds
24. Add 500 µL of 2.7 Molarity (M) - 2 Mass Percent 
pH 9 to the mixture

SECTION: Alumina extraction of catecholic compounds
25.   

SECTION: Alumina extraction of catecholic compounds
26. 20000 rcf, 10 min 
SECTION: Alumina extraction of catecholic compounds
27. Remove the aqueous layer by aspiration and wash alumina with 1 mL of Milli-Q water

SECTION: Alumina extraction of catecholic compounds
28. 20000 rcf, 10 min 
SECTION: Alumina extraction of catecholic compounds
29. Remove the aqueous layer by aspiration and wash alumina with 1 mL of Milli-Q water

SECTION: Alumina extraction of catecholic compounds
30. 20000 rcf, 10 min 
SECTION: Alumina extraction of catecholic compounds
31. Remove the aqueous layer by aspiration and wash alumina with 1 mL of Milli-Q water

SECTION: Alumina extraction of catecholic compounds
32. 20000 rcf, 10 min 
SECTION: Alumina extraction of catecholic compounds
33. Remove the aqueous layer by aspiration 
SECTION: Alumina extraction of catecholic compounds
34. Elute catechols from alumina with 100 µL  of 0.4 Molarity (M) by shaking for 2 min

SECTION: Alumina extraction of catecholic compounds
35. Collect all liquid into the injection plate without taking alumina
 

SECTION: Alumina extraction of catecholic compounds
36. Finally, inject 7 µL  into the UPLC-MS/MS system.
SECTION: UPLC-MS/MS analysis for catecholaminergic, serotonergic and cholinergic determination
38. 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.
SECTION: UPLC-MS/MS analysis for catecholaminergic, serotonergic and cholinergic determination
39. 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.
SECTION: Sample preparation
11. Take 55 µL  of sample for ACh determination:  dilute 1:4 with 0.1 % volume  containing a final concentration of 100 nM of ACh-4d as IS.

SECTION: UPLC-MS/MS analysis for catecholaminergic, serotonergic and cholinergic determination
37. 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). 
SECTION: UPLC-MS/MS analysis for catecholaminergic, serotonergic and cholinergic determination
37.1. 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.
SECTION: UPLC-MS/MS analysis for catecholaminergic, serotonergic and cholinergic determination
37.2. 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.
SECTION: UPLC-MS/MS analysis for catecholaminergic, serotonergic and cholinergic determination
37.3. 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. 
SECTION: Preparation of calibration curves
3.1. Serially dilute acetylcholine (ACh) standard with 0.1 % volume to obtain a calibration curve covering a range of 0.2-1000 nM
SECTION: Preparation of calibration curves
5.1. For ACh calibration curve, distribute the sample into 90 µL aliquots prior to the addition of 30 µL of the working mixture and 120 µL of 200 nanomolar (nM) in 0.1% FA in ACN. 
SECTION: UPLC-MS/MS analysis for catecholaminergic, serotonergic and cholinergic determination
38.1. 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. 
SECTION: UPLC-MS/MS analysis for catecholaminergic, serotonergic and cholinergic determination
40. Multiple Reaction Monitoring (MRM) acquisition settings for the targeted metabolites are
summarised in the following Table
 
 
 
SECTION: Data analysis and representation
41. 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. 
SECTION: Data analysis and representation
42. Catechol oxidation is measured using the formula AC+5SCDA+5SCDA-PB/DA + 5SCD+5SCD-PB/L-DOPA
SECTION: Data analysis and representation
43. DA synthesis is measured using the formula DA+NE+DOMA+VMA+ 3MT+DOPAC /L- DOPA
SECTION: Data analysis and representation
44. DA degradation is measured using the formula DOPAC+3MT/DA
SECTION: Data analysis and representation
45. Data is normalised by the protein concentration (determined by BCA) and presented as the percentage of the wt concentration or ratio.