I am very new to understanding UPLC, and need some help to get started. I would like to run analysis on some blood samples (I have serum and plasma) to detect concentrations of a number of hormones associated with bone metabolism (osteocalcin, pyridinoline, Vit D and K, PTH etc). Firstly, would UPLC be better for this analysis than ELISA (which is what I have been doing, but it is extremely cost prohibitive, a huge inhibitor to my area of research)? Secondly, what do I need to organise in order to get started, to save on some lab tech costs? I have determined the molecular weights of the hormones I would like to analyse. Do I need to source and locate standards? Is there a better standard to use over another? I apologise if this is very basic, as I mentioned, I am very new to this area of analysis (but very keen to understand!)
Hi Anita,
I also recently conducted UPLC analysis on plasma samples to determine relative concentrations of plasma proteins. This can be achieved label free (without standards) by carrying out MRM analysis if you are coupled to a QqQ mass spec system? See http://tmpl.uab.edu/MRMPath/ to determine peptides and Skyline software was able to compute collision energies and estimated optimal transitions. I was able to use very little sample prep (SPE plate~£200) but with proteins such as osteocalcin it may not be possible to assess multiple proteins. Obviously if you want to do a quantiative study you will need to order standards but these can be expensive too. If you would like anymore advice I can certainly send you some publications which I found helpful.
Best wishes
Hi, Anita -
Ruth Ann's advice is good; take her up on the offer of some related iterature. What I'd add, though, is to get familiar and confident with the system with standards before you begin working with actual samples. The standards don't need to be those of your analytes if these are expensive or not so easy to obtain; a set of similar structures would be fine. Make sure you can get reproducible chromatography. Determine recoveries and limits of detection in buffers and then in serum and/or plasma.
Basically, make sure you can accurately analyze known mixtures before you begin working with unknowns.
Best of luck!
Regards.
Pete
Thank you all for your advice. Ruth Ann, I would be very grateful for some publications. My email is [email protected]
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