I am going to measure dopamine in cell supernatant by HPLC method based on this paper entitked "Self-Assembling Peptide Nanofiber Scaffolds Enhance Dopaminergic Differentiation of Mouse Pluripotent Stem Cells in 3-Dimensional Culture". I asked the authours but didi not get any response from them. I do not know how can I prepare supernatant for HPLC? I have to acetylate it or not?
It depends on your sample matrix and the expected concentration of dopamine. I would perform an online search and in particular methods for dopamine on websites for LC columns (www.phenomenex.com) and HPLC methods (Waters and Agilent).
Thank you so much Pro. Neagle.
I am wanting to compare a drug treated dopaminergic cell (BE2M17 cell line) with control group. I think the amount of dopamine is about 10 microgram/ml in control group. So, do you think I have to manipulate cell supernatant?
MS detector, I would say no. Most methods use an ECD (electrochemical detector) and non-volatile buffers (a BIG no no for MS). I also think you have overestimated your expected concentrations since most articles report pg/mL (a million times less).
An example;
http://tools.thermofisher.com/content/sfs/brochures/110827-AB129-HPLC-Norepinephrine-MicrodialPerfus-16Jun2011-LPN2861.pdf
Dear Prof. Neagle,
Thank you so much for your kindly consideration. In fact, it has been measured by other laboratory last time and it was around 10 microgram/ml but they did not tell me their procedure!.
Perhaps you can use an MS, but you will have to develop a 'new' HPLC method. In a past life I validated per ICH Q2(r1) an HPLC method for epinephrine (similar for dopamine) using a reverse phase C18 column (5 cm long) for a 'clean' drug product.
Use a 5 cm C18 column, mobile phase 50/50 Methanol/Water adjusted to pH 3 with 0.1% folic acid (a volatile buffer), flow rate is 1 mL/minute. 99% Dopamine raw material can be purchased from Sigma-Aldrich. Baseline separation should occur after 5-10 minutes. Other proteins may 'gum up' your MS detector (SPE?). Always syringe filter (0.1 um) your samples. Ultrafilter your mobile phases (0.1 um)
SPE of dopamine! Great minds think alike! (Fools seldom differ)
http://www.waters.com/webassets/cms/library/docs/720005094en.pdf
Thank you so much for taking your time and help. I will try it. I hope it works.
Not dopamine, but I am sure some of your colleagues can use the information;
http://agilent.com/cs/library/applications/5991-8007EN.pdf?utm_content=sf109750041&utm_campaign=Agilent%20Technologies&utm_medium=spredfast&utm_source=linkedin&sf109750041=1
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