Hi, I am new to the test, but I am the first one to use the HPLC-ECD machine in my department to analyze plasma catecholamine.

I have 2 requests :

First, how do you prepare a standard curve. At the moment I used expired plasma as the diluent. I need to know in detail how to handle that e.g temperature of plasma.

Second, kindly review my method for desorption. Is there an alternative to sodium bisulphate?

CATECHOLAMINE ABSORPTION

1. Plasma is thawed at room temperature.
2. In a 2 mL tube, 1.2 ml of plasma is combined with 40 無 of 3,4-DHBA (internal standard).
3. The sample is shaken for 30 seconds and centrifuged for 1 minute.
4. Then sample is added with:
a. 8 mg of activated alumina
b. 320 無 of [ 2 mol/L Tris / 20g/L EDTA (pH 8.7) ]
5. Sample is shaken for 15 minutes.
6. Sample is centrifuged at 9000 g for 1 minute (microcentrifuge).
7. The supernatant is discarded and the alumina retained.
8. The alumina is washed with 800 無 of [ 0.2% Tris/EDTA (pH8.1) ]
9. It is shaken for 1 minute and centrifuged for 1 minute.


CATECHOLAMINES DESORPTION

1. Add:
a. 100 無 of acetic acid
b. 50 無 of 5% EDTA
c. 50 無 of 10% sodium bisulfate

2. 100 無 of this solution is added to the alumina and shaken for 15 minutes.
3. Centrifuge for 1 minute.
4. The supernatant with the desorbed catecholamine is filtered through a 0.045 然 syringe filter.
5. 20 無 is injected to HPLC column.

Thank you.