Hi everyone.
I am trying to develope an analytical method by HPLC based on reverse phase separation (C18 column) for determine a drug in a oftalmic presentation. The problem is that in this presentation there is a high concentration of an excipient, specifically polyvinyl alcohol (PVA) ([PVA] = 15 mg/mL) .
When I make an injection on the LC system pressure rises significantly. I think that PVA is not compatible with my mobile phase (MeOH 75% : H20 25%(acidified with AcOH to pH 3.25) and this incompatibility manifests rising pressure system.
I´ve made macroscopic studies about solubility of the excipients and no precipitation, flocculation or another kind of unstability phenomenom appears (or can see).
What type of mobile phase can use for this problem, based on my assumption of incompatibility of the PAV?
Thx.
PVA is a thickening agent and water soluble polymer, so it will thicken the mobile phase after injection. Can you do some cleanup to separate it from your analytes? Check if you can use molecular weight cut-off filter (Amicon is one of the supplier). Put the liquid sample in the vial with filter, centrifuge, your sample should be small enough to pass thru the membrane, PVA should stay in the filter.
You should try some extraction procedure. It will keep away the matrix effect. Furthermore, the sample treatment could concentrate the analytes and you will get more sensibility on analytical signal. Try liquid extraction or solid phase extraction.
good luck
If you analyte is relatively non-polar, extraction with solvent will be good and quick. Can you tell me more about the nature of your target compounds?
Ok, Thank you all.
Narong, the composition of the oftalmic presentation is:
Excipients: Hidrogenated and polyoxiethylenated Castor Oil (is not a problem)
Dehydrated alcohol (is not a problem)
Polyvinyl alcohol 14 mg/mL
Benzalkonium Chloride 0.05 mg/mL
Low concentration of NaCl, Na2HPO4, NaH2PO4, HCl or NaOH (pH reg.), dissodium edetate, H2O,
Active Drug: TACROLIMUS 300 ug/mL.
Patrick, I tried to dissolve a little part of sample in a mobile phase composed by ACN like organic modifier, and a precipitiation phenomenon appears. This macroscopic phenomenon not appears when mobile phase is composed by MeOH (but pressure rises).
I think I´ll try to filtrate the sample throw an AMICON filter of 30000 nominal molecular weight, because PAV have a molecular weight about 89000-98000.
By the other side, i´ll try to precipitate PAV with different agents, like in plasma samples for deproteinization procedures.
Any idea more?
I read about solubility in EtOH. I think would be better precipitate PVA with acetone. Solubility in acetone is lower than EtOH. In fact PVA does not have solubility in this organic solvent.
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