Techniques for disrupting protein binding in sample preparation

If you are frustrated with low recoveries caused by protein binding during Supported Liquid Extraction (SLE) and Solid Phase Extraction (SPE), this blog post will give you a few tips. Proper sample pre-treatment is the key to solving this issue.

What is protein binding?

Well, many of the drugs we commonly take and analyze for are protein bound in plasma. A drug can exist in two forms, bound and unbound depending on the drugs affinity for plasma protein. Common blood proteins that drug bind to are human serum albumin, lipoprotein, glycoprotein, and α, β‚ and γ globulins.

Since albumin is alkalotic, acidic and neutral drugs will primarily bind to albumin. Basic drugs will bind to the acidic alpha-1 acid glycoprotein.

With both SPE and SLE it is important to disrupt the protein bound drug complex to free the drug and allow it to be extracted with the best possible recovery.

Drug-protein binding should be disrupted during sample pre-treatment. These interactions can often be overcome by adjustment of the pH or by precipitating the plasma proteins. In some cases, protein-bound drugs can be effectively extracted without pre-treatment if passage through the sorbent is sufficient to disrupt the binding.

Protein binding and SLE methods

With an SLE method, to disrupt protein binding of hydrophobic analytes in serum or plasma- simply dilute the sample (1:1) (v/v) using water/isopropanol (50:50, v/v). This can disrupt the binding without causing full precipitation of proteins.

Protein binding and SPE methods

With an SPE method sample pre-treatment often is limited to the type of sorbent being used. A pH adjustment for ion exchange needs to consider the effects on protein binding as well as the proper charge state needed to load and bind to the sorbent. Sample pH may require readjustment before loading. Some strategies that can be used to disrupt nonspecific protein binding include:

2% disodium EDTA, 2% formic acid, 2% TCA, 2% Acetic acid, 2% TFA, 2% phosphoric acid and Acetonitrile (protein precipitation)

Some analytes, like for example the fat-soluble vitamins, need quite unusual extraction conditions due to the high degree of protein binding. To know more, click on the button below and download the application note.

Download the Application Note