We are performing a pancreatic lipase inhibition assay using para nitro phenyl butyrate as the substrate and Orlistat as the standard in the range of 9-150ug /total mL. Contrary to what we were expecting, we are getting the least absorbance at the lowest conc of Orlistat (0.270) and the highest absorbance (0.779) with higher concentrations of orlistat.
We have extracted orlistat from orlistat capsule mix using DMSO.
Pancreatic lipase was prepared using deionized water and
Para nitro phenylbutyrate was prepared in acetonitrile.
It's probably due either to some colored material in the orlistat capsule extract, or to some insoluble material from it.
Instead of extracting orlistat from a formulated capsule, which contains multiple substances you should buy the pure substance.
Thanks for the quick response. Yes we should have used the pure substance but it was available with our vendor only in kg quantities, hence just to initially check qualitatively if the assay was working (inhibition by orlistat) we extracted the formulation. However that was probably a bad idea. Thanks!!
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