TITLE: Determination of free and protein-bound DA and NE and their metabolites and oxidation products by UPLC-MS/MS method
AUTHORS: miquel.vila

[DESCRIPTION]
Protocol for the determination of free and protein-bound DA and NE 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 three mixtures: MIX1 (DA, L-DOPA, NE, 3MT, AC),  MIX2 (DOMA, DOPE, DOPAC) and MIX3 (5SCD and 5SCDA).
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 or MIX3), 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
11. Take 240 µL for metabolite determination and add 26 µL of IS  
 
 

SECTION: Sample preparation
12. 20000 rcf, 10 min, 4 °C 
 

SECTION: Sample preparation
13. Transfer supernatant to an Ostro protein precipitation and phospholipid removal plate (Waters, USA) to be filtered.
SECTION: Reductive HCl hydrolysis of resulting pellets
15. 
 

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
14. Inject  7 µL of filtered supernatant samples into the UPLC-MS/MS system
SECTION: Reductive HCl hydrolysis of resulting pellets
16. 20000 rcf, 10 min, 4 °C 
SECTION: Reductive HCl hydrolysis of resulting pellets
17. 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
18. Purge tubes with a stream of nitrogen, seal them and heat them at 110 °C for960 min 

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

SECTION: Reductive HCl hydrolysis of resulting pellets
20. 20000 rcf, 10 min, 4 °C 
SECTION: Reductive HCl hydrolysis of resulting pellets
21. Treat the supernatant with with acid-washed alumina to extract catecholic compounds
SECTION: Alumina extraction of catecholic compounds
22. 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
23. Add 500 µL of 2.7 Molarity (M) - 2 Mass Percent 
pH 9 to the mixture

SECTION: Alumina extraction of catecholic compounds
24.   

SECTION: Alumina extraction of catecholic compounds
25. 20000 rcf, 10 min 
SECTION: Alumina extraction of catecholic compounds
26. Remove the aqueous layer by aspiration and was alumina with 1 mL of Milli-Q water

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

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

SECTION: Alumina extraction of catecholic compounds
31. 20000 rcf, 10 min 
SECTION: Alumina extraction of catecholic compounds
32. Remove the aqueous layer by aspiration 
SECTION: Alumina extraction of catecholic compounds
33. Elute catechols from alumina with 100 µL  of 0.4 Molarity (M) by shaking for 2 min

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

SECTION: Alumina extraction of catecholic compounds
35. Finally, inject 7 µL  into the UPLC-MS/MS system.
SECTION: UPLC-MS/MS analysis
36. 
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.


SECTION: UPLC-MS/MS analysis
37. 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
SECTION: UPLC-MS/MS analysis
38. 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.