Hi,

I'm new in this area and I am a pioneer in this assay in my departmental lab. I am assaying catecholamines with HPLC-ECD. My problems are:

1. I get the peaks of Adrenaline and Noradrenaline, but their bases are touching each other. How can I seperate the bases?

2. I could not get the peak for my internal standard (DHBA). I used 400 pg of DHBA. What amount is adequate?

3. My results are not consistent. I suspect they are due to my catecholamine desorption techniques, where I used acetic acid in the end. Any suggestion of a better desorbent?

Thank you

Questions
What is your column?
What is your mobile phase?
What are your ECD conditions?
Do you think that the inconsistancy is due to your chromatography, or maybe the sample prep.

Answers
1:Different column, or mobile phase, or flow rate, or temperature, or a combination of all or some.
2:If you can't see 400 pg on column of DHBA, something is VERY wrong. You should easily see 40 pg on column, and even 4 pg should be relativly straight forward.
3:Sort out your chromatography first, then sort out the rest.

Thanks Anon,

My column is 18-ODS
My mobile phase would be:
1. Sodium acetate anhydrose
2. Heptane sulphonic acid
3. EDTA and acetonitrile
pH 3.15 titrated with acetic acid
ECD : Guard Cell +300mV, EC 1 +175 and EC 2 -175

The methods over the literatures vary too much!

If you have personally do the assay, kindly e-mail me!!

Thanks

Jane

Dear Jane,
You can consider our method as alternative to yours. Our method does not require ion-pairing reagents and uses simple mobile phase. All peaks a well resolve. Would you please check this links:
http://allsep.com/makeChr.php?chr=Chr_042 (for DOPA, Dopamine, Tyrosine and epinephrine)

http://allsep.com/makeChr.php?chr=Chr_044 (for the whole passway of phenylalanine, tyrosine, DOPA, Dopamine, Norepinephrine and epinephrine)

Contact us at (847) 229-2629 or via email at mail@allsep.com if you need more information. You can move all peaks in any direction by changing amount of acid or acetonitrile in the mobile phase. If you go with 4.6x150 or 250 mm column you will have even more room between peaks. The beauty of the first method that is short with full resolution of peaks.

Jane,

Several companies offer complete kits for running the catecholamine methods, from sample prep to HPLC. The kits are fool proof, designed for the clinical laboratory, and analysis of urine or plasma.

I just checked - plenty of names: Chromsystems, Recipe, ClinChem, ESA.

Thank you AllsepTech for your tips in changing the peaks. I'll consider your method.

Thanks Uwe Neue, but the kits are quite expensive, aren't they?

Jane

Jane,
Here is another method for the separation of your critical pair (epinephrine and norepinephrine)

http://www.allsep.com/makeChr.php?chr=Chr_045

Jane,

Indeed, validated kits for clinical analysis are expensive. But at least some of them come with a column tested for the assay, pre-prepared and validated solvents, sample preparation tools and instructions, guard columns etc. If you buy all these materials individually, you may end up very quickly close to the cost of the assembled test, and you still need to develop the separation...

Plus, you got nobody to complain too, if the separation does not work any more tomorrow :-)...

PS: I should have said that I do not belong to any of these companies nor do I hold stocks - to make clear that my recommendations are not biased...

Uwe Neue