Chromatography Forum

Can somebody tell what is the mechanism of adding HCl (Hydrochloric acid) into plasma before liquid-liquid extraction? I am studying phenylbutazone in plasma. Some references suggest adding HCl to plasma before extraction (use dicloromethane). From my study, recovery rate with adding HCl is much lower than that without adding HCl, why? Many thanks.

HCl protonates acidic drugs to their neutral forms (phenylbutazone is one) which makes them more soluble in organic solvents than the ionized salts. Given the pKa of ~4.5, you only need to lower the pH to ~3. As to why it is not working for you, is hard to say.

Thanks, Mark. After adding HCl, pH dropped to 3 from 7. Apparently I could see protein precipitation happened. In plasma, most phenylbutazone binds to protein. Is that possible that the decrease of recovery rate is due to the precipitation of protein? thanks

The addition of a large amount of acid is commonly used to destroy the protein structure and to prevent the binding of analyte to the protein. You may be dancing in an intermediate range, where the amount of acid is enough to make the analyte neutral, but not enough to destroy the protein structure. You may actually gain by increasing the amount of HCL until you reach pH 1.

There are obviously a lot of compounds which adhere to proteins (or any other solids like glass) if their solubility is not high enough in the liquid phase, regardless of the proteins structural state. Also, since proteins precipitated there is the possibility to even loose unbound analyte if any liquid phase remains with the protein after "separation" of the two phases.

You should be carefull with using HCl and phenylbutazone. This compound has a tendency to degradation. In our lab we use acetonitrile in extraction of phenylbutazone detemination in plasma . It gives perfect precipitation of proteins.

Thanks for all inputs. I am going to test the effect of different HCl volumes on the extraction rate and will poster results later.