UPLC-MS/MS analysis of dextromethorphan-O-demethylation kinetics in rat brain microsomes

Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Barent N DuBois, Reza Mehvar

Abstract

Formation of dextrorphan (DXT) from dextromethorphan (DXM) has been widely used to assess cytochrome P450 2D (CYP2D) activity. Additionally, the kinetics of CYP2D activity have been well characterized in the liver microsomes. However, studies in brain microsomes are limited due to the lower microsomal content and abundance of CYP2D in the brain relative to the liver. In the present study, we developed a micro-scale enzymatic incubation method, coupled with a sensitive UPLC-MS/MS assay for the quantitation of the rate of DXT formation from DXM in brain microsomes. Rat brain microsomes were incubated with different concentrations of DXM for various times. The reaction was stopped, and the proteins were precipitated by the addition of acetonitrile, containing internal standard (d3-DXT). After centrifugation, supernatant (2 μL) was injected onto a UPLC, C18 column with gradient elution. Analytes were quantitated using triple-quadrupole MS/MS with electrospray ionization in positive ion mode. The assay, which was validated for accuracy and precision in the linear range of 0.25 nM to 100 nM DXT, has a lower limit of quantitation of 0.125 fmol on the column. Using our optimized incubation and quantitation methods, we were able to redu...Continue Reading

Citations

May 13, 2020·Drug Metabolism Reviews·Ana Rita Silva, Ricardo Jorge Dinis-Oliveira
Nov 6, 2018·Xenobiotica; the Fate of Foreign Compounds in Biological Systems·Barent N DuBoisReza Mehvar
Apr 29, 2021·Environmental Science & Technology·Gabrielle P BlackThomas M Young
Aug 14, 2021·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Devaraj Venkatapura ChandrashekarReza Mehvar

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