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Influence of Phenobarbital on Morphine Metabolism and Disposition:LC-MS/MS Determination of Morphine (M) and Morphine-3-Glucuronide (M3G) in Wistar-Kyoto Rat Serum, Bile, and Urine

[ Vol. 8 , Issue. 1 ]

Author(s):

Yazen M. Alnouti, Melinda K. Shelby, Chuan Chen and Curtis D. Klaassen   Pages 79 - 89 ( 11 )

Abstract:


A simple LC-MS/MS method has been developed and validated for the simultaneous determination of morphine (M) and morphine-3-glucuronide (M3G) in rat serum, bile, and urine. Deuterated D3-M and D3-M3G were used as internal standards (IS) for M and M3G, respectively. Serum samples were processed by acetonitrile precipitation. Bile samples were prepared by solid-phase extraction (SPE) using Oasis MCX cartridges. Urine samples were directly analyzed after dilution with mobile phase. Chromatography was performed using a Luna C18 column (5 μm, 150 x 2.1 mm I.D.). The mobile phase consisted of acetonitrile (ACN) and 7.5 mM ammonium formate (pH 9.3) delivered from separate pumps with a simple gradient. The method was validated to quantify M in the range of 1-1000 ng/ml in bile and serum, and 0.025-25 μg/ml in urine. M3G was quantified in the range of 1-1000 ng/ml in serum, 0.1-100 μg/ml in bile, and 0.05- 25 μg/ml in urine. The method was applied to study the pharmacokinetics and disposition of M and M3G in Wistar-Kyoto (WKY) rats, and the effect of phenobarbital (PB) on M and M3G disposition. M is metabolized to M3G at a lower rate in male than female rats leading to higher M levels and lower M3G levels in serum, urine, and bile of male than female rats. PB administration induces M glucuronidation to M3G in male, but not female WKY rats, and abolishes the gender differences in M and M3G pharmacokinetics.

Keywords:

HPLC, mass spectrometry, morphine, morphine glucuronide, UGT, Wistar-Kyoto rats, phenobarbital, induction, CAR

Affiliation:

Department of Pharmacology,Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA.



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