To make:
Use source solution
Volume of source solution (mL)
Volume of CLRW
Metanephrine concentration (ng/mL)a
Normetanephrine concentration (ng/mL)a
Std 5
Spiked from Stock
160 μL
QS to 100 mL
1600.0
1600.0
Std4
Std 5
50
QS to 100 mL
800.0
800.0
Std3
Std 4
50
QS to 100 mL
400.0
400.0
Std2
Std 3
50
QS to 100 mL
200.0
200.0
Std1
Std 2
25
QS to 100 mL
50.0
50.0
2.
Controls—Lyphochek Quantitative Urine Control, Level I and II (BioRad)
Prepare control levels 1 and 2 by adding 10 mL of CLRW to each vial. Allow the controls to stand for at least 15 min, swirling occasionally.
3.
Testmix—Pipette 10 μL of Metanephrine and Normetanephrine standard stock (1 mg/mL) and 10 μL each of Metanephrine and Normetanephrine internal standard stocks (1 mg/mL) into a 50 mL Class A volumetric flask. Dilute to volume with 100 mM Ammonium Formate + 2 % Formic Acid. Stable for 1 year when stored at <−60 °C.
2.5 Analytical Equipment and Supplies
1.
Thermo Fisher Scientific Quantum Access Tandem Mass Spectrometer.
2.
Thermo Fisher Scientific CTC-PAL Autosampler and Aria TLX2 HPLC.
3.
Atlantis T3 analytical column (3 μm, 3.0 × 100 mm).
4.
T3 guard column cartridge (3 μm, 2.1 × 10 mm).
5.
T3 guard column holder.
6.
Varian Plexa SPE cartridges (30 mg, 3 mL).
7.
Injection Vials with caps (12 × 32 mm A/S Crimp, 350 μL).
8.
Pipette Tips (up to 1000 μL capacity).
9.
Pipette Tips (10–100 μL capacity).
10.
Eppendorf Combitips (25 mL total capacity).
11.
16 × 100 mm disposable borosilicate tubes.
12.
13 × 100 mm disposable borosilicate tubes.
13.
pH meter.
14.
Vortex Genie Mixer.
15.
Boiling water bath.
16.
Vacuum manifold.
17.
50 °C water bath with nitrogen supply.
3 Methods
3.1 Stepwise Procedure
1.
Turn on 56 °C and 100 °C water baths.
2.
Bring calibrators, controls, and patient samples to room temperature and mix thoroughly.
3.
To labeled 16 × 100 mm tubes, pipette 1.0 mL of calibrator, control, or patient sample.
4.
Add 100 μL internal standard to each tube.
5.
Using a plastic transfer pipette, add two drops of 3.6 M HCl to each tube. Vortex briefly.
6.
Place tubes in a metal rack and cover the top of each tube with a 16 × 125 mm Teflon-lined test tube cap to prevent evaporation. Place the covered tubes into a boiling water bath (100 °C) for 30 min to hydrolyze the samples.
7.
Transfer the tubes to a cool tap water bath. Allow the samples to cool to room temperature.
9.
Insert one labeled Plexa SPE cartridge for each sample into the vacuum box.
10.
Wash each cartridge with 1 mL of methanol. Repeat.
11.
Wash each cartridge with 1 mL 0.5 M NH4Cl. Repeat.
12.
Pour the entire hydrolyzed sample into respective Plexa SPE cartridge.
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