Page 10 - Shimadzu Journal vol.3 Issue3

Drug Discovery

Protocol Analytical method

Incubation conditions A LC-MS/MS method is developed for each compound. In addition, a
• Test Compound concentration: 10 µM calibration curve is established to quantify the amount of compound
• Microsomal Protein concentration: 0.5 mg/mL during metabolism. Two approaches are used.
• Incubation time: 60 min • Disappearance of the parent ion mass over a period of time
• Co-factors added: NADPH • Accumulation of the metabolites over a period of time
• Internal standard: Verapamil
Different SCAN and SIM modes of the quadrupoles useful in the analysis
• Positive control: HZ-166
• Q3 Scan
• Negative control: without microsomes
• Selective Ion Monitoring
• Time points: 0,10,20,30,40,50 and 60 min
• Product ion scan
• Instrument: Digital Dry bath
• MRM Transition
• Temperature: 37°C
Microsomal Assay mixture
incubated at 37°C
Compound Internal
standard
Intensity
Ice cold Acetonitrile
with Verapamil

Sonication followed by
centrifugation Retention time

Centrifuge filter tubes

Analysis by LCMS 8040

66