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how to seperate plasma interference from drug peak....
Posted: Thu Apr 16, 2009 7:17 am
by shantaram
I am confronting a problem of plasma front merging with my analyte peak.
Chromatographic conditions are:
mobile phase: MeOH:25mM phosphate buffer(38:62)
flow rate: 0.450mL/min
column: Genesis C18(100cm*4.6i.d.) 4µparticle size
oven: 40°c
Rt of my drug is 5.4....
Posted: Thu Apr 16, 2009 7:47 pm
by tom jupille
The usual suspects:
- change the aqueous/organic ratio
- change the temperature
- change the organic solvent from MeOH to ACN (or THF)
- change the pH
- change the column
There is no magic; you have to be systematic and persistent!
Posted: Fri Apr 17, 2009 9:51 am
by shantaram
The usual suspects:
- change the aqueous/organic ratio
- change the temperature
- change the organic solvent from MeOH to ACN (or THF)
- change the pH
- change the column
There is no magic; you have to be systematic and persistent!
I had tried most of them except ACN and temperature. What about temperature should i increase to how much????
Posted: Fri Apr 17, 2009 3:40 pm
by tom jupille
What about temperature should i increase to how much????
I have no idea.
Try something and see what happens.
Posted: Fri Apr 17, 2009 5:48 pm
by Kazimierz
Dear Shantaram,
You analyse intermediate lipohilic drug in serum. The front is more polar.
Therefore suggestion is:
1. Extraction plasma with methyl t. buthyl ether or methylene chloride or
ethyl acetate (manipulate with pH).
2. Column of 10 cm is much short for plasma, 25 cm or 15 cm is need.
3. I underrstand that peak symetry is good for plasma-free standard.
Posted: Sat Apr 18, 2009 6:08 am
by shantaram
Dear Shantaram,
You analyse intermediate lipohilic drug in serum. The front is more polar.
Therefore suggestion is:
1. Extraction plasma with methyl t. buthyl ether or methylene chloride or
ethyl acetate (manipulate with pH).
2. Column of 10 cm is much short for plasma, 25 cm or 15 cm is need.
3. I underrstand that peak symetry is good for plasma-free standard.
I had tried with t. butyl methyl ether and ethyl acetate, but i will look for the larger column.
Thank you
Posted: Sun Apr 19, 2009 3:29 am
by mohan_2008
From your HPLC conditions - it seems that the drug elutes fairly early and maybe closer to the dead volume.
I would try a Phenyl column to see if it improves retention - at lower temperature. Next, try a polar embed phase and last resort will be HILIC.
Posted: Sun Apr 19, 2009 5:54 am
by shantaram
From your HPLC conditions - it seems that the drug elutes fairly early and maybe closer to the dead volume.
I would try a Phenyl column to see if it improves retention - at lower temperature. Next, try a polar embed phase and last resort will be HILIC.
Thank you, i will try...