کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8942689 | 1645111 | 2018 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
UPLC-MS/MS analysis of dextromethorphan-O-demethylation kinetics in rat brain microsomes
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
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 (2â¯Î¼L) 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 0.25â¯nM to 100â¯nM DXT, has a lower limit of quantitation of 0.125â¯fmol on the column. Using our optimized incubation and quantitation methods, we were able to reduce the incubation volume (25â¯Î¼L), microsomal protein amount (5â¯Î¼g), and incubation time (20â¯min), compared with reported methods. The method was successfully applied to estimation of the Michaelis-Menten (MM) kinetic parameters of dextromethorphan-O-demethylase activity in the rat brain microsomes (meanâ¯Â±â¯SD, nâ¯=â¯4), which showed a maximum velocity of 2.24â¯Â±â¯0.42â¯pmol/min/mg and a MM constant of 282â¯Â±â¯62â¯Î¼M. It is concluded that by requiring far less biological material and time, our method represents a significant improvement over the existing techniques for investigation of CYP2D activity in rat brain microsomes.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography B - Volume 1096, 1 October 2018, Pages 66-72
Journal: Journal of Chromatography B - Volume 1096, 1 October 2018, Pages 66-72
نویسندگان
Barent N. DuBois, Reza Mehvar,