This Application Note describes how the BioTrove RapidFire high-throughout mass spectrometry (RF-MS) system interfaced to an Agilent 6410 triple quadrupole mass spectrometer was used for the characterization of the enzymatic reaction of CYP450 3A4/5 enzyme in a preparation of human liver microsomes and for the determination of IC50 curves of poten- tially inhibiting drug compounds.
This document details an ultra-high throughput method for determining CYP450 enzyme inhibition by combining an Agilent 6410 triple quadrupole mass spectrometer with the BioTrove RapidFire RF-MS inlet system. The approach aims to match the speed of fluorescence-based assays while providing biologically relevant data using liver microsomes instead of recombinant enzymes.
The note outlines the experimental protocol for characterizing CYP 3A4/5 enzyme kinetics and screening drug compounds. It specifies the equipment used, including MassHunter workstation software and BioTrove RapidFire integrator software, along with detailed MRM settings and incubation conditions.
| Total sample to sample time | 7 seconds (including autosampler overhead) |
|---|---|
| Incubation temperature | 37 °C |
| Injection volume | 10 pL |
| Wash flow rate | 1.5 mL/min |
| Elution time | 3s |
The system uses an Agilent 6410 triple quadrupole mass spectrometer interfaced with a BioTrove RapidFire RF-MS inlet system.
Data acquisition uses Agilent MassHunter workstation software, while final data analysis is performed using BioTrove RapidFire integrator software.