Automated high throughput human CYP isoform activity assay using SPE-LC/MSmethod: application in CYP inhibition evaluation

1. A high throughput screening (HTS) method for the evaluation of the seven major human hepatic CYP isoform activities was developed on a 96-well form at, with automation. The method utilized pooled human liver microsomes and seven probe substrates, generic conditions for incubation, reaction termina tion and metabolite extraction with solid phase extraction (SPE) plates. Me tabolites from the seven reactions were pooled and quantified using a gener ic liquid chromatography and tandem mass spectrometry (LCMS/MS) method. 2. The HTS method was validated based on K-m values obtained, which were in agreement with literature data. 3. The isoform inhibition profiles of ketoconazole, quinidine, sulfaphenazo le, tranylcypromine, alpha-naphthoflavone, and 4-methylpyrazole against CYP s 3A4, 2D6, 2C9, 2A6 (and 2C19), 1A2 and 2E1, respectively, were obtained b y this HTS method. Graphically obtained IC(50)values are in agreement with literature reported values. 4. The HTS method represents a significant efficiency and selectivity impro vement over traditional methods, and can be used for CYP inhibition assay a nd can be extended for liver activity profiling.