how to seperate plasma interference from drug peak....

[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....

[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!

[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????

[tom jupille]

What about temperature should i increase to how much????

I have no idea. Try something and see what happens.

[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.

[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

[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.

[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...