How to prepare the mobile phases that are used in application note 720005094EN: Rapid and Simultaneous Analysis of Plasma Catecholamines and Metanephrines Using Mixed-Mode SPE and Hydrophilic Interaction Chromatography for Clinical Research - WKB101140
OBJECTIVE or GOAL
Prepare the mobile phases and sample preparation reagents that are used in application note 720005094EN.
ENVIRONMENT
- Plasma catecholamines
- Plasma metanephrines
- Solid-phase extraction (SPE)
- Bridged ethylene hybrid (BEH)
- Amide
- Hydrophilic interaction chromatography (HILIC)
- Xevo TQ-S
- Clinical research
PROCEDURE
Preparation of pH 3.0 mobile phases for catecholamine/metanephrine analysis
- Equipment and Reagents
1.1. pH meter capable of 3-point calibration
1.2. Calibration buffers at pH 1.68, 4.0, and 7.0
1.3. Ammonium formate, 99%; Acros or equivalent. MW = 63.06
1.4. Formic acid, 99% LC/MS grade
1.5. Ammonium acetate, 99%
1.6. Acetonitrile, LC/MS grade
- Preparation of 400 mM ammonium formate stock
2.1. Calibrate pH meter.
2.1.1. Calibrate the pH meter at pH 1.68, 4.0, and 7.0. Many pH meters are calibrated at pH 4.0, 7.0 and 10.0. For accurate reading at pH 3.0, it is necessary to extend the calibration range. A pH 1.68 buffer is commonly available as a standard and is usually one of the default calibration values in most commercial pH meters.
2.2. Weigh out 12.6 g ammonium formate.
2.3. Dissolve in approximately 300 mL MilliQ water (18.0 MΩ). Stir until fully dissolved. Sonicate for at least 10 minutes to ensure complete dissolution.
2.4. Add 15-25 mL pure formic acid (Optima) until pH reaches 3.0.
NOTE: Ensure that the pH meter is calibrated. Once pH 3 is reached, allow the solution to mix for 10 minutes. After 10 minutes, check the pH reading again. Adjust to pH 3.0 as required.
2.5. Q.S. to 500 mL with MilliQ water.
NOTE: Ammonium formate and formic acid are volatile solvents and will evaporate from solution, giving this solvent a limited shelf life. Please evaluate the stability of this critical component.
- Preparation of Mobile Phase B (MPB): 85:15 ACN:H2O w/ 30 mM NH4COOH pH 3.0 (Plasma Method)
3.1. 75 mL of 400 mM ammonium formate stock (pH 3.0)
3.2. 75 mL MilliQ water
3.3. 850 mL acetonitrile (Optima)
3.4. Sonicate for 10 minutes to fully dissolve the mobile phase. (This salt content (30 mM) is on the border of dissolving in 85% ACN, so it must be sonicated. However, once in solution, it will stay in solution and not require additional manipulation.) Note: This is a critical component of the assay. If made incorrectly, poor peak shapes will result, especially for the catecholamines. If poor peak shape is observed, do not hesitate to remake this mobile phase.
- Preparation of Mobile Phase A (MPA): 5:95 ACN:H2O w/ 30 mM NH4COOH pH 3.0
5.1. 75 mL of 400 mM ammonium formate stock (pH 3.0)
5.2. 875 mL MilliQ water
5.3. 50 mL ACN (Optima)
- Preparation of Needle Washes
6.1. Fixed Loop (FL) system: Place WNW and SNW in MPB.
6.2. Flow through needle (FTN) system:
6.2.1. Purge: 85:15 ACN:H2O
6.2.2. Wash: Place in MPB
Preparation of sample preparation reagents
50 mM ammonium acetate: 0.771 g qsp 200 mL water (Plasma Method)
1.1. Alternatively, prepare by diluting 0.5 M stock 1:10 in MilliQ water.
NOTE: Prepare daily, as 50 mM ammonium acetate buffer can develop algal growth.
- 20 mM acetate ammonium: 0.308 g qsp 200mL water
2.1. Alternatively, prepare by diluting 0.5 M stock 1:25 in MilliQ water.
NOTE: Prepare daily, as 20 mM ammonium acetate buffer can develop algal growth.
- 85:15 ACN:H2O: 170mL ACN + 30 mL water
- 85:15 ACN:H2O + 2%FA: 50 mL of 85:15 ACN:H2O. Add 1 mL formic acid.
NOTE: Prepare daily.