Chromatography Forum

Hi,

We are having a problem transferring methods from HPLC with UV/Vis detection to MS detection. We are trying to analyse drug concentrations in mouse brain (and liver and blood). In the past, we used a homogenisation buffer containing Brij 35 as prepare the tissue before solid-phase extraction of the drugs.

However, we see a large background signal when we analyse the same samples using LCMS, we think this is due to the Brij 35 detergent.

Can anyone suggest an alternative recipe for a homogenisation buffer?
Is the detergent really necessary?
Are there sample prep products available for removing detergents?

Many thanks,

Philip

Why does your buffer contain Brij35? Is the step following homogenisation the solid phase extraction? In that case, the choice of buffer or other 'liquid' will depend on the drug and the SPE procedure you use.

regards Bert

We used a literature reference (analysis of doxycycline in arterial plaques) to choose our homogenisation buffer recipe, outlined below. The Brij 35 is presumably to break up cell membranes?

We homogenise, spin down to remove solid matter, then perform SPE using Waters Oasis cartridges. We were attracted to SPE since we are processing large numbers of samples with limited manpower.

Homogenisation buffer recipe:

Urea 2M, Tris 50mM, NaCl 1g/l, EDTA 1g/l, Brij 35, Phenylmethanesulphonyl fluoride 0.02 0.1mM, pH 7.6.

Philip

For homogenisation of the tissue, you should choose a liquid inwich your analyte is extractable and will stick to yourSPE column. The liquid can be a simple buffer, but also water or fys. saline, all depending on the characteristics of the drug. We use for homogenisation always an ultra-turrax. For break up of cell membranes, you can also place your samples in an ultrasonic bath before SPE.

Hope this helps,

regards Bert