Simultaneous determination of midazolam, dextromethorphan and omeprazole rat plasma by LC-MS

Aim: To develop an LC-MS method for the simultaneous determination of midazolam, dextromethorphan and omeprazole in rat plasma. Methods: Afte raddition of 10 μL of diazepam solution (5 μg/mL, internal standard) to 0.1 mL plasma and alkalization with 100 μL of 0.2 mol/L Na₂CO₃, plasma was extracted with 5 mL of ether. 3 mL of organic layer was then transferred and evaporated to dryness. The residue was reconstituted in 200 μL methanol and 5 μL of aliquots was injected into an ODS C₁₈ (250 mm × 2.0 mm, 5.0 μm) column. The mobile phase consisted of 0.01% NH₄OAc-methanol(30:70) at a flow rate of 0.2 mL/min. The elution from the HPLC column was plumbed directly into ESI probe. Analysis in the mass spectrometer was operated in the selected ion monitoring mode. The mass spectrometers was operated in SIM m/z: 326.0 for midazolam, m1z: 272.1 for dextromethorphan, m/z: 346.0 for omeprazole, and m/z: 284.9 for diazepam. Results: The lowest limit of quantitation of midazolam, dextromethorphan and omeprazole in rat plasma was 2.0 ng/mL, with good recovery of above 85% and good precision which was expressed as intra-day and inter-days relative standard derivation below 10%. Conclusion: The established LC-MS is suitable for pharmacokinetic study of midazolam, dextromethorphan and omeprazole and could be applied in high through-put screen of new drugs as "cocktail" research.