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HPLC method for brain amines asssay.
Posted: Sun Mar 08, 2009 7:22 pm
by khalidrauf
Hi
I am working on brain neurotransmitters isolated from straitum, nucleus acumbens etc.I have a a shim pack C18 column with electrochemical detector. coulchem iii with 5011 A analytical cell and 5020guard cell.I am interested in analysis dopmine, dopac HVA, 5HT,and noradrenaline.Please tell me some method thats gives result with my current instruments set up in one chromatogram.please give details of cells potentials applied and mob phase conditions.
Thanks.
Posted: Tue Mar 10, 2009 4:51 pm
by paraboxa
We analyse catecholamines and 5HIAA in 24 hour urine samples. Both involve an extraction step (cation exchange on SB-HEMA for Cats, di-ethyl ether for 5HIAA).
Cats: phsophate mobile phase, C18 column. Detector is 5011A analytical cell with 5021A guard (Coulochem II). We run it in reductive mode, gain on auto between 500nA and 1000nA. Our program switches from 500nA after the internal standard is eluted.
Much the same for the 5HIAA detector settings. 5HIAA also uses aqueous phosphate but this time a Spherisotb ODS2 column.
I can give you more detail if you wish. HTH
Posted: Wed Mar 11, 2009 10:16 am
by khalidrauf
Dear Paraboxa.
Thanks indeed for taking time and such a usefull reply.I shall highly appreciate further detailed input from you on the subject.Sir i have got method on ESA coulchem site for simultaneous determination of six neurotransmitters and metabolites.I have never used ECD and have a little superficial concepts regarding these nano amperes range.I have set 100 u amperes without any knowledge regarding this.Please update me on the significance of the nanao amperes as the literature i have studied speaks only of E1 and E2 voltage n guard cell potential.Please also tel me whats reductive mode.or recommend some literature.I m using guard cell potential 340, E1 -150 and E2 +200.Additioanlly my mob phase is 90mM sod di hydrfen ortho phosphate buffer with 50mM Cittric acid and1.7mM 1 octane sulfonic acid and 50uM EDTA.flow rate 0.6 ml.checked dopamine only standard in 0.1 m perchloric acid bt no peak.
I am sure you shall help m e out,
with warmest regards
Khaid Rauf
Ph.D Scholar